A salient question is whether (…) the agreement permits or prohibits arbitration on a classwide basis. Here we must answer a question one step removed—who decides whether the agreement permits or prohibits classwide arbitration, a court or the arbitrator? The question has divided the many state and federal courts to consider it.
We conclude no universal rule allocates this decision in all cases to either arbitrators or courts. Rather, who decides is in the first instance a matter of agreement, with the parties‘ agreement subject to interpretation under state contract law. Under state law, these parties‘ arbitration agreement allocates the decision to the arbitrator. Under federal arbitration law, no contrary presumption requires a different result, so the issue remains one for the arbitrator.
The issue before us is not whether class arbitration is permissible here, but a matter antecedent to that issue: who should decide whether it is permissible, a court or an arbitrator. No universal one-size-fits-all rule allocates that question to one decision maker or the other in every case. Rather, who decides is a matter of party agreement. As the United States Supreme Court has explained in a closely related context, just as the arbitrability of the merits of a dispute depends upon whether the parties agreed to arbitrate that dispute, so the question who has the primary power to decide arbitrability turns upon what the parties agreed about that matter. (First Options of Chicago, Inc. v. Kaplan (1995) 514 U.S. 938, 943.) And just as whether class arbitration is available depends on whether the parties agreed to allow or forbid it (Stolt-Nielsen S. A. v. AnimalFeeds Int’l Corp., supra, 559 U.S. at pp. 684–687), so the question who has the power to decide the availability of class arbitration turns upon what the parties agreed about the allocation of that power.
The United States Supreme Court‘s treatment of the issue confirms the parties‘ agreement as the mandatory starting point. In Green Tree Financial Corp. v. Bazzle (2003) 539 U.S. 444 (Green Tree), the plurality and the principal dissent, although disagreeing about the ultimate who decides question, both agreed about where the analysis should begin. (See id. at p. 451 (plur. opn. of Breyer, J.) [concluding the question should be for the arbitrator because under the terms of the parties‘ contracts, the question—whether the agreement forbids class arbitration—is for the arbitrator to decide]; id. at p. 456 (dis. opn. of Rehnquist, C. J.) [agreeing that the decision of what to submit to the arbitrator is a matter of contractual agreement by the parties].) Similarly, in Stolt-Nielsen S.A. v. AnimalFeeds Int’l Corp., supra, 559 U.S. at page 680 and Oxford Health Plans LLC v. Sutter (2013) 569 U.S. ___, ___, fn. 2 [186 L.Ed.2d 113, 119, fn. 2, 133 S.Ct. 2064, 2068, fn. 2], the Supreme Court accepted—because the parties had so agreed—that an arbitrator should decide in the first instance whether class arbitration was available.
The parties do not disagree as to which state‘s law applies: California.
These features of the arbitration clauses suggest the who decides question is an arbitrable one, but they are by no means conclusive. In the presence of ambiguity, we turn to other principles applicable to the interpretation of arbitration clauses and contracts generally.
Ultimately dispositive here are two other long-established interpretive principles. First, under state law as under federal law, when the allocation of a matter to arbitration or the courts is uncertain, we resolve all doubts in favor of arbitration. (Wagner Construction Co. v. Pacific Mechanical Corp. (2007) 41 Cal.4th 19, 26; Engalla v. Permanente Medical Group, Inc. (1997) 15 Cal.4th 951, 971; see Mastrobuono v. Shearson Lehman Hutton, Inc. (1995) 514 U.S. 52, 62 & fn. 8.) All else being equal, this presumption tips the scales in favor of allocating the class arbitration availability question to the arbitrator.
As Lebo Automotive asserts, City of Los Angeles confirms the strong presumption that courts should determine the jurisdiction of arbitrators. (See City of Los Angeles v. Superior Court, supra, 56 Cal.4th at p. 1096; Code Civ. Proc., § 1281.2 [on a petition to compel arbitration, the court should order arbitration if it determines that an agreement to arbitrate the controversy exists].) That is what we, a court, are tasked with doing here; consistent with the presumption, we are engaged in deciding whether a decision on the availability of class arbitration is for a court or an arbitrator.
(To the extent Keating and its progeny hold a court may order class arbitration based on the interests of justice and without regard to the parties‘ wishes (see Keating v. Superior Court, 31 Cal.3d at pp. 613–614), they are no longer good law (see Stolt-Nielsen S.A. v. AnimalFeeds Int’l Corp., 559 U.S. at p. 684 [a party may not be compelled under the FAA to submit to class arbitration unless there is a contractual basis for concluding that the party agreed to do so]).
This is not a case where the parties by agreement have expressly opted for the application of state law in lieu of any otherwise applicable federal rule. In the absence of such an opt out, the issue whether the availability of class arbitration is a question of arbitrability subject to a pro-court presumption arises in the shadow of federal law. Any state law presumption, were there one, would have to yield to whatever presumption the FAA establishes. We thus turn to federal law.
The crux of this case is whether the FAA imposes an interpretive presumption that, as a matter of federal law, preempts state law rules of contract interpretation and alters the conclusion state law would otherwise reach here. Congressional intent is the touchstone of any preemption analysis (e.g., Quesada v. Herb Thyme Farms, Inc. (2015) 62 Cal.4th 298, 318; Brown v. Mortenson (2011) 51 Cal.4th 1052, 1060), but neither the FAA‘s text nor its legislative history speaks to the question we face or offers insight as to that intent. Accordingly, we look to judicial understandings of the FAA.
However, as the United States Supreme Court has twice reiterated in cases not directly addressing the who decides question, Green Tree contains no controlling view concerning what presumption, if any, the FAA requires when interpreting the parties‘ agreement as to who decides class arbitration availability. (See Stolt-Nielsen S.A. v. AnimalFeeds Int’l Corp., 559 U.S. at pp. 678–679; Oxford Health Plans LLC v. Sutter, 569 U.S. at p. ___, fn. 2 [186 L.Ed.2d at p. 119, fn. 2, 133 S.Ct. at p. 2068, fn. 2].) The issue thus remains unresolved. While accepting that extant Supreme Court cases do not decide the matter (dis. opn., post, p. 7), the dissent argues that from what they do decide, it is only a small leap to the conclusion that the availability of class arbitration is a question to presumptively withhold from the arbitrator (id. at p. 9). In the absence of controlling authority, however, we must resist the urge to take that leap without convincing evidence the FAA imposes an anti-arbitral presumption that overrides the state law reading of the parties‘ arbitration clause. In our view, the FAA does not.
As the Green Tree plurality recognized, the availability or unavailability of class arbitration has nothing to do with whether the parties agreed to arbitrate, either in general or with respect to a specific dispute. Instead, the question is of the what kind of proceeding sort that arises subsequent to the gateway issue of whether to have an arbitral proceeding at all.
We disapprove Garden Fresh Restaurant Corp. v. Superior Court, supra, 231 Cal.App.4th 678, to the extent it is inconsistent with this opinion. We also disapprove Kinecta Alternative Financial Solutions, Inc. v. Superior Court, supra, 205 Cal.App.4th 506, to the extent it suggests the availability of class arbitration is always an issue for the court.
Lebo Automotive argues that state law requires harmless error review in all cases before reversal will follow. (Cal. Const., art. VI, § 13; Code Civ. Proc., § 475.) Not so; some errors are reversible per se. The error here falls within that class requiring automatic reversal because its effects are unmeasurable and defy analysis by harmless-error standards. (People v. Blackburn (2015) 61 Cal.4th 1113, 1135; see People v. Collins (2001) 26 Cal.4th 297, 311; Martin v. County of Los Angeles (1996) 51 Cal.App.4th 688, 698.) We cannot say whether an arbitrator would have decided the issue the same or differently. Indeed, to deny remand by insisting an arbitrator would surely have agreed with the trial court‘s view or our view of the merits of class availability is to recommit the very error complained of—deprivation of a decision by a contractually agreed-upon decision maker. The denial of the parties‘ right to their agreed-upon decision maker is thus the sort of miscarriage of justice that requires reversal without further harmless error analysis.
Secondary sources: Am. Arbitration Assn., Supplementary Rules for Class Arbitration (2003) rule 3 [allocating the class availability question to the arbitrator]; JAMS, Class Action Procedures (2009) rule 2 [same].
(Cal.S.C., July 28, 2016, Sandquist v. Lebo Automotive, Inc., S220812).
Arbitrage de classe en Californie, la décision de l’autoriser revient-elle au Tribunal ou à l’arbitre ? Notion d’erreur dans les considérants sans effet notable sur le dispositif.
Une question importante posée par cette affaire est celle de savoir si la clause compromissoire convenue par les parties au contrat permet ou prohibe un arbitrage de classe (le contrat de base était un contrat préformé de près de 100 pages entre un vendeur de voitures et son employeur ; ce contrat est susceptible de concerner de nombreux vendeurs dans la même situation). Mais la présente espèce pose surtout une question en amont, à savoir qui décide si le contrat permet ou prohibe un arbitrage de classe : s’agit-il du Tribunal ou de l’arbitre ? Cette question a divisé les nombreuses cours étatiques et fédérales qui l’ont considérée.
En résumé, la Cour Suprême de Californie juge en l’espèce qu’aucune règle absolue ne permet de trancher cette question. Bien plutôt, la réponse est donnée par le contrat lui-même, interprété selon le droit étatique applicable. En l’espèce, selon le contrat d’arbitrage, la décision est de la compétence de l’arbitre. Et selon le droit fédéral de l’arbitrage, aucune présomption contraire ne vient contredire cette conclusion.
De même que l’arbitrabilité de la question au fond posée par un litige dépend de la question de savoir si les parties ont convenu ou non d’arbitrer dit litige, de même la question de savoir qui dispose de la compétence de décider d’un arbitrage dépend de ce que les parties ont convenu à ce niveau.
La Cour doit dès lors examiner l’accord des parties pour déterminer ce qui est exprimé s’agissant de la question de « qui décide ». Mais à la lumière de quel droit ? Fédéral exclusivement ou dans un premier temps étatique puis fédéral ? En effet, pour décider si les parties se sont mises d’accord pour arbitrer une certaine dispute, les Tribunaux appliquent généralement les principes ordinaires du droit de l’état régissant la formation des contrats. Cette règle de conflit par défaut s’applique à la question de savoir si la disponibilité ou non de l’arbitrage de classe est tranchée par l’arbitre ou par le Tribunal, en tant que cette question de savoir « qui décide » présente un point litigieux d’interprétation des contrats : le droit étatique régit en priorité ce type de situations.
Les parties s’accordent à soutenir que le droit californien s’applique ici, et non celui d’un autre état.
En interprétant une clause d’arbitrage, la Cour considère les expectatives des parties s’agissant de l’allocation des responsabilités. Typiquement, les parties qui concluent une clause d’arbitrage s’attendent à ce que leur litige soit résolu sans qu’il soit nécessaire de solliciter le Tribunal. Dans les nombreux cas où les parties s’accordent à saisir l’arbitre et commencent la procédure en soumettant leurs mémoires à l’arbitre, exiger que la question « qui décide » (d’un arbitrage de classe) relève de la compétence du Tribunal contreviendrait aux attentes des parties et imposerait des coûts et des délais additionnels substantiels : les parties devraient tout d’abord suspendre la procédure arbitrale, puis saisir le Tribunal de leur litige relatif à l’interprétation de la clause arbitrale, éventuellement recourir ensuite contre la décision du Tribunal, et enfin seulement retourner devant l’arbitre pour la suite de la procédure arbitrale. En l’espèce, la clause d’arbitrage mentionne une préférence pour l’arbitrage du fait de ses coûts réduits et de son efficacité particulière.
De plus, la règle exigeant de résoudre les ambiguïtés à l’encontre de la partie qui a rédigé s’applique avec une force particulière dans le cas d’un contrat d’adhésion, où la partie qui rédige dispose d’un pouvoir de négociation supérieur, tandis que l’autre partie ne dispose pas de la force économique pour modifier le texte du contrat.
Ce principe général d’interprétation des contrats s’applique également à l’interprétation des clauses d’arbitrage.
Aucune des parties en l’espèce ne conteste le principe, bien établi en droit californien aussi bien qu’en droit fédéral, selon lequel les parties en litige peuvent convenir de conférer à l’arbitre la compétence de décider si un litige particulier relève ou non de l'arbitrage. En l’absence d’accord des parties dans ce sens, cette décision revient au Tribunal.
La jurisprudence Keating v. Superior Court, 31 Cal.3d at pp. 613–614, en tant qu’elle attribuait au Tribunal la compétence d’ordonner un arbitrage de classe fondée sur l’intérêt de la justice et sans égard à la convention des parties, est renversée. La présente affaire consiste donc en un revirement de jurisprudence à ce niveau.
En l’espèce, la convention des parties n’a pas expressément opté pour l’application du droit étatique à la place du droit fédéral. En l’absence d’un tel « opt out », la question de savoir si la disponibilité d’un arbitrage de classe relève du concept de base d’arbitrabilité, présumé de la compétence du Tribunal, s’analyse à la lumière du droit fédéral. Que dit ce droit à cet égard ?
Il est déterminant d’analyser si la loi fédérale sur l’arbitrage impose une présomption d’interprétation constituant préemption des règles étatiques d’interprétation des contrats et altérant les conclusions qui découleraient du droit étatique. L’intention du Congrès est le point de départ de toute analyse en matière de préemption. Or ni le texte de la loi fédérale sur l’arbitrage ni son histoire législative ne se prononcent à ce niveau, ni n’offrent d’éléments qui pourraient être utiles. Dès lors, la Cour se tourne vers la jurisprudence appliquant la loi fédérale sur l’arbitrage.
La Cour Suprême fédérale s’est directement prononcée sur notre question « qui décide » à une reprise (Green Tree, 539 U.S. 444) : dans cette affaire, la convention des parties soumettait à l’arbitre toutes les disputes, prétentions ou controverses, qui naissaient du contrat ou qui se trouvaient liées au contrat, ainsi que les relations qui résultaient du contrat. La pluralité des Juges a interprété ce langage comme conférant à l’arbitre la compétence de se prononcer sur la disponibilité de l’arbitrage de classe. Par ailleurs, les mêmes quatre Juges ont expliqué que rien dans la loi fédérale sur l’arbitrage ne soumettait la question « qui décide » à une présomption contraire en faveur d’une compétence du Tribunal. Par conséquent, la pluralité des Juges a conclu que cette question d’interprétation des contrats relève de la décision de l’arbitre, et non du Tribunal.
Cependant, comme la Cour Suprême fédérale l’a indiqué dans la jurisprudence Stolt-Nielsen S.A. v. AnimalFeeds Int’l Corp. et Oxford Health Plans LLC v. Sutter, qui ne concernaient pas directement la question « qui décide », la décision Green Tree ne contient pas d’opinion ayant valeur de précédent s’agissant de savoir quelle présomption éventuelle la loi fédérale sur l’arbitrage requiert en interprétant l’accord des parties s’agissant de la question de « qui décide » de la possibilité ou non de recourir à un arbitrage de classe. Le problème reste ainsi non résolu. In casu, la Cour Suprême de Californie n’entend pas retenir que la loi fédérale sur l’arbitrage imposerait dans cette discussion une présomption contre l’arbitrage qui l’emporterait sur l’interprétation par le droit étatique de la convention d’arbitrage. Et la Cour de poursuivre en jugeant que la loi fédérale sur l’arbitrage n’impose rien de cette sorte.
La Cour Suprême fédérale a interprété la loi fédérale sur l’arbitrage comme imposant deux présomptions distinctes, en relation avec les prétentions de fond. D’une part, les Tribunaux ont à présumer que l’intention des parties est de confier auxdits Tribunaux la compétence de trancher, en cas de litige à ce sujet, la question de l’arbitrabilité, soit la question de savoir s’il existe une convention d’arbitrage exécutoire, ou si cette convention s’applique au litige concerné.
Et les Tribunaux présument que les parties entendent que ce soient les arbitres, et non la justice ordinaire, qui décident des litiges concernant le sens et l’application de préconditions procédurales particulières, préconditions devant être remplies avant de permettre la mise en œuvre de la procédure arbitrale. Même la remise en cause de l’exécution de l’entier du contrat qui contient la clause d’arbitrage est présumée relever de la compétence de l’arbitre.
La disponibilité ou non d’un arbitrage de classe est sans rapport avec la question de savoir si les parties se sont mises d’accord ou non sur le principe d’un arbitrage, soit de manière générale, soit relativement à une difficulté spécifique. Au contraire, cette disponibilité ou non est une question subséquent à la question seuil de savoir si la procédure d’arbitrage est ou non retenue.
La Cour renverse ici deux précédentes jurisprudences en ce qu’elles disposaient que la question de la mise en œuvre ou non d’un arbitrage de classe relevait toujours de la compétence du Tribunal et non de l’arbitre (Garden Fresh Restaurant Corp. v. Superior Court, supra, 231 Cal.App.4th 678 ; Kinecta Alternative Financial Solutions, Inc. v. Superior Court, supra, 205 Cal.App.4th 506).
S’agissant de la question de l’erreur sans effet notable sur le dispositif du Jugement (« harmless error ») : l’une des parties soutient que le droit californien impose de revoir dans chaque cas une erreur dans les considérants sous l’angle de « harmless error » (évitant ainsi une annulation du Jugement inférieur si l’erreur dans les considérants est sans effet notable sur le dispositif) (Cal. Const., art. VI, § 13; Code Civ. Proc., § 475). Tel n’est pas le cas, juge en l’espèce la Cour Suprême de Californie. Certaines erreurs entrainent une annulation automatique du Jugement inférieur. L’erreur commise dans la présente affaire est de cette sorte, car ses effets ne sont pas mesurables et ne peuvent pas valablement être analysés sous l’angle de « harmless error ». la Cour ne peut pas dire si un arbitre se serait en l’espèce prononcé de la même manière ou différemment. Et ne pas annuler le Jugement inférieur en jugeant qu’un arbitre se serait sûrement prononcé de la même manière que l’autorité inférieure s’agissant de la disponibilité d’un arbitrage de classe revient à commettre l’erreur de priver du pouvoir de décider celui qui avait été contractuellement nommé à cet effet, soit l’arbitre.
Kirk is a qualified automotive expert witness with experience in complex business litigation, economic damages calculations, business valuation, and settlement warranty extension valuation.


text:
Mr. Proctor Knott Owens for plaintiff in error. [241 U.S. 340, 341] Mr. Grant Fellows, Attorney General of Michigan, and Mr. David H. Crowley for defendant in error.
The general purpose of the act is well expressed in its title-'An Act to Provide for the Licensing, Bonding, and Regulation of Private Employment Agencies, the Limiting of the Amount of the Fee Charged by Such Agencies, the Refunding of Such Fees in Certain Cases, the Imposing of Obligations on Persons, Firms, or Corporations Which Have Induced Workmen to Travel in the Hope of Securing Employment, Charging the Commissioner of Labor with the Enforcement of This Act, and Empowering Him to Make Rules and Regulations, and Fixing Penalties for the Violation Hereof.' It provides: Sec. 1. No private employment agency shall operate without a license from the commissioner of labor, the fee for which is fixed at $25 per annum except in cities over two hundred thousand population, where it is $100; this license may be revoked for cause; the commissioner is charged with enforcement of the act, and given power to make necessary rules and regulations. Sec. 2. A surety bond in the penal sum of $1,000 shall be furnished by each applicant. Sec. 3. Every agency shall keep a register of its patrons and transactions. Sec. 4. Receipts containing full information regarding the transactions shall be issued to all persons seeking employment who have paid fees. Sec. 5. 'The entire fee or fees for the procuring of one situation or job and for all expenses, incidental thereto, to be received by any employment agency, from any applicant for employment at any time, whether for registration or other [241 U.S. 340, 343] purposes, shall not exceed 10 per cent of the first month's wages;' no registration fee shall exceed $1 and in certain contingencies one half of this must be returned. Sec. 6. 'No employment agent or agency shall send an applicant for employment to an employer who has not applied to such agent or agency for help or labor;' nor fraudulently deceive any applicant for help, etc. Sec. 7. No agency shall direct any applicant to an immoral resort, or be conducted where intoxicating liquors are sold. Sec. 8. Violations of the act are declared to be misdemeanors and punishment is prescribed.
The supreme court of Michigan held 'the business is one properly subject to police regulation and control;' the prescribed license fee is not excessive; provisions of the state Constitution in respect of local legislation are not infringed; and no arbitrary powers judicial in character are conferred on the commissioner of labor. But it did not specifically rule concerning the validity of limitations upon charges for services specified by 5.
Considering our former opinions it seems clear that without violating the Federal Constitution a state, exercising its police power, may require licenses for employment agencies, and prescribe reasonable regulations in respect of them, to be enforced according to the legal discretion of a commissioner. The general nature of the business is such that, unless regulated, many persons may be exposed to misfortunes against which the legislature can properly protect them. Williams v. Fears, 179 U.S. 270, 275 , 45 S. L. ed. 186, 189, 21 Sup. Ct. Rep. 128; Gundling v. Chicago, 177 U.S. 183, 188 , 44 S. L. ed. 725, 728, 20 Sup. Ct. Rep. 633; New York ex rel. Lieberman v. Van De Carr, 199 U.S. 552, 562 , 563 S., 50 L. ed. 305, 310, 311, 26 Sup. Ct. Rep. 144; Kidd, D. & P. Co. v. Musselman Grocer Co. 217 U.S. 461, 472 , 54 S. L. ed. 839, 845, 30 Sup. Ct. Rep. 606; Engel v. O'Malley, 219 U.S. 128, 136 , 55 S. L. ed. 128, 136, 31 Sup. Ct. Rep. 190; Rast v. Van Deman & L. Co. 240 U.S. 342, 365 , 60 S. L. ed. --, 36 Sup. Ct. Rep. 370; Armour & Co. v. North Dakota, 240 U.S. 510, 513 , 60 S. L. ed. --, 36 Sup. Ct. Rep. 440; See Moore v. Minneapolis, 43 Minn. 418, 45 N. W. 719; Price v. People, 193 Ill. 114, 55 L.R.A. 588, 86 Am. St. Rep. 306, 61 N. E. 844; People ex rel. Armstrong v. Warden, 183 N. Y. 223, 2 L.R.A.(N.S.) 859, 76 N. E. 11, 5 Ann. Cas. 325. In its general scope, and so far as now [241 U.S. 340, 344] sought to be enforced against plaintiff in error, the act in question infringes no provision of the Federal Constitution. The charge relates only to the plainly mischievous action denounced by 6. Provisions of 5 in respect of fees to be demanded or retained are severable from other portions of the act, and, we think, might be eliminated without destroying it. Their validity was not passed upon by the supreme court of the state, and has not been considered by us.

matches: v. v. v. v. v. v. v. v. v. v.


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Cryptochromes and photolyases form a flavoprotein family in which the FAD chromophore undergoes light induced changes of its redox state. During this process, termed photoreduction, electrons flow from the surface via conserved amino acid residues to FAD. The bacterial (6–4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Mutagenesis studies have identified Trp342 and Trp390 as essential for charge transfer. Trp342 is located at the periphery of PhrB while Trp390 connects Trp342 and Tyr391. The role of Tyr391, which lies between Trp390 and FAD, is however unclear as its replacement by phenylalanine did not block photoreduction. Experiments reported here, which replace Tyr391 by Ala, show that photoreduction is blocked, underlining the relevance of Tyr/Phe at position 391 and indicating that charge transfer occurs via the triad 391-390-342. This raises the question, why PhrB positions a tyrosine at this location, having a less favourable ionisation potential than tryptophan, which occurs at this position in many proteins of the photolyase/cryptochrome family. Tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings. Since replacement of Tyr391 by Trp resulted in loss of FAD and DMRL chromophores, electron transfer cannot be studied experimentally in this mutant, but calculations on a mutant model suggest that Trp might participate in the electron transfer cascade. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad. Our results suggest that evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent.
The conservation of Tyr or Trp for electron transfer shows that this selection is not a random process. Factors such as the chemical environment (other nearby amino acids or water) certainly play a significant role. Electron transferring Tyr residues are usually located close to the protein surface, surrounded by water and/or located close to deprotonating amino acids.15,16 Electron transferring Trp residues can occur at the periphery or in the centre of a protein.
The group of FeS-BCP proteins is a phylogenetically distinct group of (6–4) photolyases (Fig. 1) with unique properties such as an Fe–S cluster and a 6,7-dimethyl-8-ribityllumazine (DMRL) antenna chromophore.17 The ET in FeS-BCP proteins differs from all other cryptochrome and photolyase groups in several ways. In the FeS-PCB members PhrB from Agrobacterium fabrum (PDB 4DJA)17 and CryB from Rhodobacter sphaeroides (PDB 3ZXS)18 photoreduction proceeds via Trp390 and Trp342 (PhrB numbering), as shown by site directed mutagenesis. These residues are highly conserved in FeS-BCP members (see ESI Fig. S1†). In both proteins, the Tyr391 side chain is directly located between Trp390 and FAD (see also Fig. 2) suggesting that this Tyr must be part of the ET chain. However, when the Tyr was replaced by Phe, the photoreduction rate of PhrB was not affected,19 and only slightly affected in CryB.18 In about 30% of FeS-BCP proteins, a Phe is placed at this position (see Fig. S1†).
– is this residue involved in photoreduction as the first electron donor of FAD, as proposed by its spatial position?
– if yes, how can the electrons be transmitted via this Tyr residue and why is it a Tyr residue, whereas in other groups of photolyases and cryptochromes a Trp residue serves as electron donor for FAD?
– Phe is usually used to interrupt electron chains. Why does the replacement of Tyr391 by Phe not interrupt the electron chain?
FAD photoreduction involving a Trp triad has been widely studied by computational approaches in Escherichia coli photolyase,21–23Arabidopsis thaliana cryptochrome,24–29Synechocystis sp. CRY-DASH protein30 or Xenopus laevis (6–4) photolyase.31 These studies highlighted the role of Trp in the classical triad27–29 or of additional residues30,31 at an atomistic level, as well as the crucial role of the environment24,25,29 or the quantum effects in these ultrafast charge transfers.26 In our group, we have established a quantum mechanics/classical mechanics (QM/MM) scheme based on fragment orbital tight-binding density functional theory (FODFTB) coupled to MM molecular dynamics (MD).22–24,32 Our approach allows direct simulations of the charge propagation along a Trp triad. Previously we established our method by successful reproductions of ET rate constants in E. coli photolyase23 and Arabidopsis cryptochrome24 and also highlighted the role of environment in the downhill charge transfer process. Applying these techniques, we are able to shed light onto the molecular evolution of ET pathways in different proteins belonging to the cryptochrome–photolyase family.
In the present work, we investigate the role of Tyr391 in PhrB by experimental and QM/MM approaches, comparing PhrB wild type (WT) with its Y391F, Y391W and Y391A mutants. We regard that the analysis of the Tyr to Trp replacement is important for an understanding of the evolution of photolyases and cryptochromes, which most often have a Trp triad as electron transmission. Due to the difference in ionization potentials, it is expected that a Tyr residue slows down or blocks charge transfer, and that the replacement of Tyr by Trp would result in increased ET rates. However, all forward and backward charge transfers along the triad and subsequent possible charge recombination with FAD have to be considered. The comparison between PhrB and other members of the cryptochrome–photolyase family shed light on protein fine-tuning, enhancement of FAD photoreduction rates and avoidance of charge recombination.
Site directed mutagenesis, protein expression and purification. A PhrB E. coli expression vector based on pET21b was used for recombinant expression of PhrB in ER2566 cells. The vectors for WT and the Y391F mutant are described in earlier publications.19,33 To obtain the Y391A and Y391W mutants, site directed mutagenesis was performed according to the Quik Change mutagenesis kit (Agilent) using a pair of complementary primers (Table S1†) with the desired mutation in the middle for initial polymerase reactions. Mutagenesis success and correctness of the sequences were confirmed by DNA sequencing. Expression and purification followed the procedure described in ref. 11 for WT and mutants. In brief, E. coli cells from agar plates were used for the inoculation of 3 l LB containing ampicillin. Following specific induction of recombinant expression with IPTG and subsequent incubation over night at 28 °C, all purification steps were carried out at 4 °C. Cells were harvested by centrifugation, suspended in 50 ml extraction buffer (50 mM Tris/HCl, 5 mM EDTA, 300 mM NaCl, 10% glycerol, pH 7.8) and extracted with a French Press (America Instrument Company) at 1000 bar. Following centrifugation and precipitation of soluble protein by ammonium sulfate (93% saturation), the protein pellet was suspended in EDTA free buffer. Soluble protein was purified by Ni affinity chromatography followed by size exclusion chromatography. The final buffer was 50 mM Tris/HCl, 5 mM EDTA, 300 mM NaCl, 10% glycerol, pH 7.8.
Photoreduction measurements by UV/vis spectroscopy. PhrB WT and mutant proteins were diluted to a final concentration of ca. 10 μM. The samples were incubated at 4 °C in darkness in saturated oxygen solution. During this treatment, reduced FADH− is converted to oxidised FAD, although spectral analyses revealed that the fraction of oxidised FAD differed among the different proteins. Thereafter, 10 mM 1,4-dithiotreitol were added to the protein solution. UV/vis spectra were recorded using a Jasco V550 photometer with temperature control adjusted to 10 °C. After the first recording, the sample was illuminated with blue light emitting diodes (λmax = 470 nm) with a light intensity of 55 μmol m−2 s−1 at the position of the cuvette. Subsequent spectra were recorded at a series of time points as given in the results section. For data evaluation, complete spectra, 450 nm or 580 absorbance values, which stand for FAD in the oxidised or semireduced forms respectively, were presented.
Cofactor detection and repair assay. For detection of FAD and DMRL, 85 μM protein was denatured by 95 °C incubation for 5 min. The insoluble protein and the soluble chromophores were separated by 15 000 × g 10 min centrifugation and 10 μL supernatant were analysed by HPLC (Agilent system with a Gemini C18 column (50 × 4.60 mm, 110 Å, Phenomenex)). The HPLC buffer conditions were: 5% acetonitrile (ACN) in 0.1% formic acid for 0–5 min; 5–75% ACN in 0.1% formic acid for 5–25 min. The flow rate was set to 0.75 ml min−1 and the column temperature to 25 °C. Elution was monitored at 260 nm and 400 nm.
The photorepair reaction mixture contained 5 μM of the purified (6–4) photoproduct of t-repair_1 (Table S1†) and 8.5 μM protein in repair buffer (50 mM Tris–HCl, pH 7.0, 1 mM EDTA, 100 mM NaCl, 5 mM MnCl2, 5% (w/v) glycerol, 14 mM 1,4-dithiothreitol). Aliquots were irradiated with 400 nm light emitting diodes (250 μmol m−2 s−1) for 3 min. Thereafter, the reactions were stopped by heating to 95 °C for 10 min. Samples were centrifuged at 15 000 × g for 10 min and the supernatants analysed by HPLC (same column and system as above). The buffer conditions were: 7% acetonitrile (ACN) in 0.1 M triethylamine acetate (TEAA) (pH 7.0) for 0–5 min; 7–10% ACN in 0.1 M TEAA (pH 7.0) for 5–35 min. The flow rate was set to 0.75 ml min−1 and the column temperature to 25 °C.
Model structures and molecular dynamics simulations. The structural model of PhrB WT has been derived from the X-ray crystal structure of Zhang et al. (PDB ID 4DJA).17 The Y391F, Y391A and Y391W mutants have not been crystallized. For the setup of the model structures, we suppose that the mutation of Tyr391 does not affect the structure of the remainder of the protein. Starting from the PhrB-WT model structure, we replaced the aromatic cycle of Tyr391 by a phenyl, a methyl or an indol ring. Two conformations of the indol ring are allowed by the protein structure, but steric hindrance prevents rotation from one to the other (see Fig. 5). In the first conformation, the Trp side chain can orient toward FAD being in a closer contact than in the second conformation. The conformations are termed Y391Wp (for proximal) and Y391Wd (for distal) in the following.
All mentioned simulations were performed with the GROMACS 5.0.4 package34,35 using the AMBER-SB99-ILDN force field.36,37 The force field parameters for neutral (oxidised) FAD and negatively charged cofactor FAD˙− were taken from riboflavin and adenosine diphosphate (ADP) models developed in previous studies.23,24 The GAFF parameters38,39 were used for the DMRL antenna chromophore. The DMRL atomic charges were calculated by restrained fitting on the electrostatic potential (RESP)40,41 at HF/6-31G* (ref. 42 and 43) level with Gaussian 09 package.44 Bonded parameters of the cubic FeS-cluster were taken from ref. 45 and the charges were taken from ref. 46.
The loop region from residues 180 to 182, which might impact the DNA binding abilities,17 was not structurally resolved in the X-ray structure of WT PhrB. It was reconstructed using the MODELLER program.47 WT and mutated proteins (Y391F, Y391A and Y391W) were solvated in a 106.24 Å3 cubic box filled by TIP3P water molecules.48 Twelve sodium ions were added to create a neutral system.
Equilibration of the solvated proteins (WT and mutants) starts with a minimization step, followed by 100 ps MD in the NVT ensemble and 100 ps in the NPT ensemble. 100 ns of production NPT MD simulations were performed afterwards. Nose–Hoover thermostat49 was used to keep a constant temperature at 300 K and Parinello–Rahman barostat50 to keep the pressure at 1 atm. Covalent hydrogen bonds were fixed on a constant length by the use of the LINCS algorithm.51 The time step for the MD simulations was 2 fs.
Site energy and electronic coupling calculations. To treat the charge transfer processes, a quantum mechanical treatment of the active site has to be included via a so called combined Quantum Mechanics/Molecular Mechanics (QM/MM) scheme. The structural part of interest for charge transfer, which is treated at QM level, contains the side chains of amino acids involved in the called triad (A, B and C, see Fig. 2) and the isoalloxazine ring of FAD. The remaining atoms are treated classically using force fields (MM) and affect the QM zone by electrostatic interactions. Hydrogen link atoms52 are inserted at the QM/MM boundary, namely in the Cα–Cβ bond of A, B and C side chains or in the C1–C2 bond of the FAD D-ribitol tail.
This FO-DFTB approach has been extensively evaluated and tested in previous publications32,54,57 and has been so far successfully applied to describe the charge transfer in photolyase,23 cryptochrome24 and DNA58,59 where the HOMO's of the fragments were used to calculate the evolution of electronic couplings and site energies.
The HOMO energies for different molecules show non-systematic errors, which can be corrected adding a constant energy shift depending on the chemical identity of the fragment. This correction was evaluated in ref. 58 for the relative energies of Trp and Tyr and expanded in this work to Phe and FAD relative to Trp (see Table S2†). The energy gap between two sites can be related to the driving force of a charge transfer as described in the Marcus theory.
The off-diagonal elements of the Hamiltonian correspond to the direct electronic coupling HDA between two sites.
These matrix elements (HDA) can be used to compute two different charge transfer regimes. When large energy barriers occur along the charge transfer pathway, tunnelling matrix elements (TDA) can be computed (‘super-exchange tunnelling’), for small barriers, a ‘direct’ hopping-type mechanism may appear, which we treat using a QM/MM non-adiabatic MD technique, also based on the same computed matrix elements HDA.
where βDA describes the direct electronic interaction between D and A, βDi and βjA the electronic interactions between D or A and the i or j-th fragment of the bridge. The tunnelling energy εtun corresponds to the average of the eigenvalues of the effective Hamiltonian and Gij is an element of the Green's function matrix, describing the probabilities for the electron to tunnel through the i–j space of the bridge.
Direct electron transfer. We established in our previous work a multi-scale method to describe charge transfer reactions in cryptochromes and photolyases.23,24 This non-adiabatic charge propagation scheme allows simulations where the charge transfer occurs on the same timescale as environment response charge state.
Nonadiabatic QM/MM MD simulations directly calculate the population of the charge which is located at a specific site of the QM zone.62 This method doesn't force a specific charge transfer process; however it can describe a hopping or even a band-like conduction.32,57 The wave-function associated with the transferred charge and atomic coordinates are simultaneously propagated solving time-dependent Schrödinger equation and classical Newton equations at every MD step, respectively. The excess charge corresponds to a second order perturbation of the neutral system Hamiltonian while environment interacts with the QM part as point charges. To take into account charge transfer, the partial charge of the QM part is calculated at every MD step to model the interaction of the moving charge with the charge distribution of the environment. More detailed description can be found in ref. 32, 57 and 62.
As for the electronic coupling calculations, charge propagation simulations were performed using an in-house GROMACS 4.6 version.63 The charge transfer between the triad and the isoalloxazine ring is initiated by the excitation of the FAD cofactor. The first transfer continuing the excitation was studied in some photolyases to happen within one picosecond.64 These two ultra-fast events were excluded in the previous studies23,24 to reduce complexity and to focus on charge transfer along the Trp triad. To keep consistency, the same exclusion was also applied here and the QM part consists in A, B and C.
Charge propagation simulations start with an electronic state where FAD˙− is a radical anion and the first site A a radical cation. The hole on the first site is then propagated along the triad and moves in the reverse direction compared to the electron. These simulations were performed on PhrB WT and the Y391W mutant. Charge occupation of each site, varying from 0 (neutral state) to 1 (fully oxidized state) is followed during 1 ns QM/MM MD simulations. We randomly chose 20 to 25 starting structures from classical MD trajectories to guarantee well equilibrated systems while sampling different initial conditions. A kinetic model23,24 was used to fit the average occupations (from the individual simulations) of each site and thus determine the different reaction rate constants.
For photoreduction studies, we generated the Y391F, Y391W and Y391A mutants of PhrB. All mutants and WT PhrB were expressed in E. coli and purified by Ni chromatography and size exclusion chromatography. Whereas protein yields of Y391F and Y391A are comparable to those of WT, the yield of Y391W is ca. 10 times lower. Absorbance spectra of Y391A and Y391F in the oxidised FAD state are comparable with WT (Fig. 3), although detailed analyses reveal different chromophore to protein ratios and/or different fractions of reduced FAD at starting time. The absorbance of the Y391W in the blue spectral range is very weak (Fig. 3). Chromophore analyses show that this mutant contains only (1.3 ± 0.1)% FAD and (1.8 ± 0.2)% DMRL as compared to WT. These values are (94 ± 1)% and (87 ± 3)% for FAD and DMRL of Y391F, respectively, and (53 ± 2)% and (53 ± 2)% for FAD and DMRL of Y391A, respectively. We propose that the replacement of Tyr 391, which is located close to FAD, by bulky Trp in Y391W results in opening of the FAD pocket and loss of FAD binding capacity. The DMRL pocket is formed by amino acids of the N-terminus and more distant from the mutation. The loss of DMRL results therefore probably from FAD depletion. The partial loss of both chromophores to equal percentages in the Y391A mutant supports this idea. The 410 nm peak in the spectrum of the Y391W mutant is assigned to the iron sulphur cluster.
Fig. 3 UV-visible spectra of PhrB WT and mutated proteins Y391A, Y391F and Y391W. Black line: without illumination; red line: illuminated by 470 nm blue light for 90 min.
During blue light irradiation, the spectra of WT PhrB and the Y391F mutant change in a characteristic manner. The transient increase at 580 nm, the maximum of the protonated FAD semiquinone, and the loss of absorbance at 450 nm, characteristic for the loss of oxidised FAD (Fig. 4), are comparable to data published earlier.33 Here, the relative A450 nm decrease at t = 90 min in the Y391F mutant appears smaller than in WT. This can be due to slower photoreduction or smaller fraction of oxidised vs. total FAD in the mutant. Both decay curves can be fitted with monoexponential decay functions which yielded time constants of 36 ± 1 min and 32 ± 1 min for WT and Y391F, respectively. Thus, the rate of overall photoreduction is not affected by the Tyr to Phe replacement, but the oxidation state of Y391F was incomplete at the start of the photoreduction experiments. Formation and decay of the semiquinone intermediate absorbing at 580 nm is slower in Y391F (rise and decay times of 6 ± 0.3 min, 110 ± 30 min for WT and 7 ± 0.3 min, 200 ± 110 min for Y391F, respectively). This results shows that the role of Tyr or Phe differs in the first and second electron transfer. In summary, the present and published data19 show clearly that the replacement of Tyr by Phe into the proposed electron path does not block photoreduction. We do not observe any light induced absorbance changes in the Y391A mutant (Fig. 3). This result suggests that position 391 is critical for photoreduction, as proposed above. DNA repair in the presence of Mn2+ (ref. 11) is complete after 5 min for WT PhrB and Y391F mutant, whereas no repair activity is observed for Y391A and Y391W mutants under these conditions. When the repair time is prolonged to 120 min, Y391W repairs about (8.7 ± 0.7) % of damaged DNA, whereas with Y391A still no repair is observed.
Fig. 4 Photoreduction of PhrB and its mutants. Absorption values at 450 nm (upper picture) were taken from UV-visible spectra measured at indicated time points upon onset of blue-light illumination. For each protein, these values were normalized against the value measured at t = 0 min. Absorption values at 580 nm (lower picture) after subtraction of the t = 0 value and normalization to the absorbance at 280 nm.
We performed 100 ns classical MD simulations of PhrB WT and the Y391F, Y391A and Y391W mutant, based on the crystal structure of PhrB. In these simulations, no major conformational change of the overall protein structures is observed. The residues A, B and C (see Fig. 2) involved in the triad occupy similar positions in WT and the Y391F or Y391A mutants. In the model structure of the Y391A mutant, water molecules fill the space let by the replacement of Tyr to Ala (see Fig. S2†). In WT, Y391F and Y391A, a water molecule, present in the crystallographic structure, interacts with isoalloxazine O4 and A backbone (see Table S3 and Fig. S3†). The experimental findings for the Y391W mutant, which has lost both chromophores, suggests a more drastic impact on the protein structure which would require simulation protocols dedicated to protein folding and FAD docking. Our simulations, which follow the dynamics of Y391W on relatively short time-scales, however, allow us to investigate the theoretical role of an amino acid independent on large structural changes that might occur on much longer time-scales. Such simulations help us to compare the relationship between environment and the first site in different members of the cryptochrome–photolyase family. In the end, we have to combine experimental and theoretical results to obtain the maximum information for the role of each amino acid. The simulations for the Y391W mutant revealed two conformations of the Trp391 side chain (A) (see Fig. 5), denominated as Y391Wd and Y391Wp. In Y391Wd, A stays continuously close to the second Trp side chain B whereas in Y391Wp, A stays most of the time close and parallel to FAD isoalloxazine ring, but moves closer to B during a few nanoseconds (see Fig. 5, S4 and S5†).
Fig. 5 Two conformations, proximal (p, upper picture) or distal (d, lower picture) of A in the Y391W mutant.
As shown in Table 1, the distances between all neighbouring sites (FAD–A, A–B, B–C) in WT and mutant structures are between 5 and 8 Å. These are typical nearest neighbour distances reported in our MD simulations of Arabidopsis cryptochrome and E. coli photolyase22–24 which allow for sufficiently large electronic couplings in order to enable fast charge transfer. The larger distances between FAD and the second neighbour between 12 and 13 Å (FAD–B) suggest that a direct electron transfer from B to FAD is unlikely because the electronic coupling decreases exponentially with distance in absence of charge transfer bridge. We also report the distance between FAD or B and another tyrosine, Tyr395 (see also Fig. 6), which is close to the charge transfer chain. Mutations have no impact on the position of Tyr395; it stays at around 8 Å from FAD and slightly less than 7 Å from B.
Fig. 6 Active site with FAD and sites A, B and C (left). Hydrogen bond network involving the hydroxyl groups of Tyr391, Tyr395, Thr373, Tyr395, Thr358, the backbone carbonyl group of Val354 and a structurally characterized water molecule (right).
Upon excitation of FAD by light or by energy transfer from the excited antenna chromophore, this site is reduced to the negatively charged FAD˙− species by an ET from the triad, which then in turn becomes positively charged. In a first step, we investigate the electronic structure of the neutral FAD–triad system by computing site energies and electronic couplings. In a second step, we compute the changes in the electronic structure due to the charge transfer. In previous work, we have studied Arabidopsis cryptochrome and E. coli photolyase in a similar way.23,24 Both proteins show a highly exergonic and fast charge transfer on a picosecond time scale. To investigate the effect of the mutation at site A, we compare the relevant parameters for charge transfer in the present study with these two reference systems.
Average values of energies and couplings are reported in Fig. 7, the error bars indicate the associated standard deviation. According to these data, site energies of Trp390 and Trp342 (B and C) are similar, independent of the chemical nature of A. The electronic coupling values associated with the charge transfer between these Trp's are about 4.1–4.2 meV for WT and the Y391F mutant, 5.9 meV for the Y391A mutant and between 7.0 and 9.0 meV for the two rotamers of the Y391W mutant. These values are comparable with those found in Arabidopsis cryptochrome and E. coli photolyase. The small increase of electronic coupling from WT to Y391W corresponds to a small decrease in B–C distance in the mutant (Table 1). For reference, we also show the location of the HOMO level of neutral FAD, which is the electron acceptor after excitation of one of its electrons.
Fig. 7 Average site energies (top, eV) and average electronic couplings (bottom, in meV) for PhrB WT and its mutants Y391F, Y391A and Y391Wp/d. Error bars denote the corresponding standard deviation. The different symbols in each site energies panel from left to right represent values for FAD (blue) and the residues A (black), B (red) and C (green). Electronic coupling HDA symbols (purple) are positioned between the two sites considered as donor and acceptor.
The site energy of A as an aromatic residue (Trp, Tyr or Phe) inside the protein follows the same order as the HOMO energies in gas phase (Table 2, HOMO ≈ −IP): HOMO(Trp) > HOMO(Tyr) > HOMO(Phe). In the protein, A has a value of about −5.8 eV for Trp, which is substantially decreased to −6.3 eV for Tyr and to around −7 eV for Phe. In the Y391W mutant, the energy of A is similar to energies of B or C, leading to a small energy gap between the charge transfer partners (within about 0.3 eV). Electronic couplings are twice as large as in PhrB WT or Y391F. These results support the possibility of a charge migration involving a Trp triad in the Y391W mutant. The distal conformation Y391Wd facilitates B → A charge transfer by decreasing the A–B energy gap by 0.08 eV and the average A–B distance by 0.64 Å compared with Y391Wp (Table 1 and Fig. 7). On the contrary, in the proximal conformation, the aromatic ring of A and the isoalloxazine ring are in a close position which must enhance A → FAD charge transfer.
As expected, the lower site energies of Tyr in PhrB WT and Phe in Y391F may present a barrier for hole transfer from FAD, as clearly seen in Fig. 7. While our previous studies on Arabidopsis cryptochrome or E. coli photolyase showed a hopping type mechanism along the Trp-triad, one may expect in WT and Y391F a tunnelling mechanism to be in operation, which would lead to less efficient charge transfer. We calculated the electronic coupling between FAD and B which involves A as a bridge for the WT and Y391F by a previously published method.54 Our calculation shows a coupling of 0.06 and 0.02 meV for WT and Y391F which is ten-fold more than the direct (assuming no bridge residue) coupling between FAD and B (see ESI Table S4†). Electron tunnelling through the A side chain seems possible, as also indicated by the experiments on Y391F. Tunnelling involving a Phe and protein backbone has been also described for the E. coli photolyase.67 Therefore, in the case of Y391F, where a barrier of more than 1 eV is apparent, we definitely have to consider tunnelling, for the smaller barrier resulting from the presence of Tyr391 in WT this is not necessarily the case. This has been discussed for charge transfer in DNA,68 where small charge transfer barriers up to 0.4 eV may easily be overcome, especially for short bridges due to molecular fluctuations. The hole transfer between FAD and A falls in this range, allowing a direct hopping mechanism (Fig. 7). Electron hopping via Tyr would involve a transiently positively charged side chain, which must be followed by deprotonation. However, a nearby proton acceptor is missing in the PhrB structure, ruling out the deprotonation mechanism of Tyr391. Oxidised Tyr cannot be stabilised while the electronic coupling between A and B is strong (see Fig. 7). If FAD → A transfer occurs, the following hole transfer between A and B must be very fast.
Other charge transfer pathways can also be considered from the crystallographic structure. A charge transfer chain via Tyr399 and Tyr40 has been suggested in our previous experimental study.19 Spectral changes related to FAD reduction in Y399F mutant are slightly slower compared to WT. However, the absence of FAD reduction in both W390F and W342 mutants clearly indicates that Trp390 and Trp342 are essential in the charge transfer process, which cannot be compensated by a transport via Tyr399 and Tyr40. Consequently, the Tyr399–Tyr40 charge transfer pathway was rejected.
Another possibility could be a transfer between FAD and Trp390 via Tyr395 (see Fig. 6). This residue could substitute site A (Tyr391), since it has similar distances to FAD and site B, as shown in Table 1. Interestingly, theses distances do not change upon mutation at site A, i.e. the FAD binding pocket and the connection via Tyr391 is very similar in all variants. According to the computed electronic couplings and site energies (Table S5†), this pathway seems to be possible as well. However, our photoreduction studies for the Y391A mutant clearly indicate the absence of any charge transfer, i.e. the pathway does not seem to be a possible alternative. Although it has been shown that our scheme for the calculation of charge transfer couplings is very reliable for all couplings between the oxidisable residues,60 the couplings between FAD and site A respective Tyr391 are very small, i.e. a small change in geometry could lead to a different result. For example, the PhrB structure in solution (experimental photoreduction measurements) could differ from the crystal structure used for calculations. Further, the couplings are computed for neutral FAD in the ground state, whereas the active state is an excited state, which may have some impact on the value of the coupling. As a consequence, within the accuracy of the calculations reported here, the rather small value reported for the FAD–Tyr395 coupling (Table S5†) could as well turn out to vanish. Therefore, we believe that under the conditions the experiments are performed, the pathway via Tyr395 seems to be impeded. However, since an analogous alternative pathway has been reported for the PhrB homologue CryB,18 it could be interesting to investigate under which conditions this pathway could be activated in PhrB. This, however, would require further investigations which are beyond the scope of the present work. For the main focus of this work, it is very interesting to note that for this alternative pathway a tyrosine is the primary electron donor of FAD, i.e. highlighting the role of tyrosine in the charge transfer cascade, which is the main emphasise of this work.
To estimate the effect of a water molecule bridging the FAD and the backbone of site A (see ESI†), we also applied the pathways model from Beratan and co-workers.69 The couplings for these pathways are very small, therefore we did not consider these pathways further (see Table S4 and Fig. S6†).
We now discuss the energetics and couplings for the case, where FAD and one of the residues of the triad are charged. The energetic landscape is changed drastically when a charged FAD and a charged A are considered (Fig. 8). As discussed recently, the fast charge transfer in cryptochromes and photolyases can be explained by a steep downhill energetics, which results from the interaction of the charge with the protein and solvent environment.23,24 The first ET results in negatively charged FAD˙− and a positively charged side chain on one site of the triad. Charge separation has a sizable effect on the site energies. The positive charge on A, B or C leads to a strong polarization of the environment. This polarized environment in turn leads to a stabilization of the charge at the respective site. In Marcus theory, this effect is called outer-sphere reorganization energy; in our simulations it manifests itself by the lowering of the site energies with regard to the neutral states. For each charge state on A, B and C, we compute the site energies along 1 ns MD simulations containing the charge on the respective site during the MD simulations, as shown in Fig. 8. We have discussed this effect in some details in our previous work, showing that the solvent has a distinct impact, in particular on those sites which are more solvent exposed,24 like site C which is located on the protein surface.
Fig. 8 Average site energies (eV) of positively charged A (black), B (red) and C (green) for PhrB, Arabidopsis cryptochrome24 and E. coli photolyase.23 Error bars denote the corresponding standard deviation. For PhrB site A, the energies labelled with Y, Wp and Wd correspond to WT, Y391Wp and Y391Wd respectively.
In previously studied Arabidopsis cryptochrome or E. coli photolyase, the positive charge stabilization follows a downhill scheme, with an increasing energy gap between neutral and charge residue from A to C and where neighbouring sites have an energy difference of about 0.5 eV (Fig. 8). This leads to the fast charge transfer in a picosecond regime.
In PhrB, a similar stabilization on C occurs, this results from the solvent exposure. However, surprisingly also site A is massively stabilized, for Tyr even more than for both Trp rotamers. In the latter, A and B site energies are similar and the charge transfer from B to A no longer follows the downhill scheme described for the WT, E. coli photolyase or Arabisopsis cryptochrome. In total, the energy difference between site C and A in Y391W is only half of the value compared to the other two systems (Fig. 8), which has a drastic effect on the charge transfer equilibrium.
There is an obvious energetic difference for the site A Trp in Y391W, E. coli photolyase and Arabidopsis cryptochrome. In Y391W, A is nearly isoenergetic to B, while in the other proteins, positively charged B is more stable than A by about 0.4 eV. In our previous work, we have analysed structural reasons for this energy gap between A and B. In the E. coli photolyase or Arabidopsis cryptochrome, A is buried in a pocket with more than 5 Å distance from any water molecule, as documented by calculation of water distribution functions along MD simulations (Fig. 9). On the contrary, water molecules can easily move toward A in PhrB: the radial distribution function of water around Tyr391 presents a peak around 5 Å, while the first peak around B is observed at 7 Å (Fig. 9). Moreover, a stable hydrogen bond network, involving Thr373, a water molecule, Tyr385, Thr358 and the backbone of Val354 (Fig. 6) can also participate in A˙+ stabilization in our simulation. In E. coli photolyase and Arabidopsis cryptochrome, sites B and C are stabilised by solvent interactions, leading to a larger solvent reorganization energy. This explains the downhill energetics as shown in Fig. 8. On the contrary, water molecules close to the FAD–A complex in PhrB help to stabilize the charge-separated RP-A (FAD˙−– A˙+) state, and compensate the unfavourable intrinsic IP of Tyr. Likewise, this could impact the charge transfer efficiency in the Y391W mutant by increasing the probability to localize the charge on the first member of the triad.
Fig. 9 Radial distribution function of water molecules around A (black), B (red) and C (green) in PhrB WT, E. coli photolyase, Arabidopsis cryptochrome. Integration of the radial distribution curves is given in insets.
Fig. 10 Time dependent evolution of the charge occupation along the triad in PhrB WT, Y391Wp mutant and Y391Wd mutant. Black line: RP-A; red line, RP-B: green line: RP-C. The graphs show the average occupation from all simulations of 1 ns length for WT or Y391W mutants respectively (see Fig. S7† for more detailed results). Error bars denote the standard deviation each 0.1 ns. The curves resulting from a fit using a kinetic model24 as described in the text are also shown using the same colours.
In WT, the population of the first radical state RP-A drops within a few ps and no back transfer is observed. The second state, RP-B, is transiently occupied by 60% of the total positive charge before decay and formation of the third state RP-C. After 25 ps simulation time, the charge distribution remains stable, with roughly 85% of the charge on the third state, while 15% remains on the second state. Due to the averaging over several simulations, these numbers show a statistical distribution rather than a charge delocalization over different sites. The positive charge is therefore well stabilized on the solvent accessible Trp342. No backward charge transfers from B to A occur during 1 ns of simulation time. The very fast first charge transfer from Tyr391 to Trp390 shows a significantly larger transfer rate than that calculated for plant cryptochrome.1 PhrB k23 and k32 are consistent with values from Arabidopsis cryptochrome and E. coli photolyase, with a backward transfer 10 fold smaller than the forward one.
The Y391W mutants show a different behaviour. On average, the hole remains on RP-A during the first 50 ps in Y391Wp and during the first 21 ps in Y391Wd, respectively. The final stabilization of 70–80% of the charge on site C occurs after 70 ps for Y391Wp and 33 ps for Y391Wd. For the A–B transfer, the forward and backward rate constants are very similar in each rotamer simulation, as shown in Table 3. In Y391Wd, the k21 rate is also close to k23. For the transfer from B to C, the forward rate is 10-fold higher than the backward rate. All rate constants are in the same order of magnitude as those of Arabidopsis cryptochrome or E. coli photolyase. The main difference between the Y391W mutant and Arabidopsis cryptochrome is the strong back transfer rate from B to A (k21 in Table 3).
Indeed, during all charge transfer simulations of our different cryptochromes and photolyases proteins, we observe several backward transfers to A. No backward transfer is present in PhrB WT simulations, as the first residue is a Tyr. We compare the number and the stability of backward transfers in Table 4 for different systems: Y391Wp/d and E. coli photolyase. In Y391W, we observe about 30 crossings in which the positive charge moves back to A and forms a stable RP-A for at least 500 fs which is ten-fold more than in E. coli photolyase. The difference between the two conformations of Y391W is related to the A–B distance: in Y391Wd, A is closer to B, which facilitates backward and forward charge transfer between them. On the contrary, in Y391Wp, A is closer to negatively charged FAD which contributes to stabilize the positive charge on A. Furthermore, a charge recombination on the isoalloxazine ring becomes more likely. Nevertheless, in 100 ns MD simulation of Y391Wp, motion of A to a distal conformation is observed and can also contribute to an enhanced charge transfer between the two Trp residues. In both conformations, the charge is more often back transferred to A, but stays less time for one transfer than in E. coli photolyase.
Like other members of the cryptochrome–photolyase family, PhrB is able to reduce FAD upon light absorption via a long range ET involving aromatic residues. Site directed mutagenesis experiments have shown that two Trp, Trp390 and Trp342, are essential for the reduction process. The distance between the isoalloxazine ring and Trp390 is roughly 12–13 Å (Table 1) and thus too far from FAD for direct ET. There is no other Trp in the structure that can complete the triad, which raises the question of the role of the closest residue to FAD involved in charge transfer.
The presence of Tyr391 is intriguing: it is situated between FAD and Trp390 in a suitable place to take part in the ET, but its oxidation appears – at first sight – not required for FAD reduction: (i) mutation of Tyr391 to redox inert Phe residue neither blocks FAD photoreduction nor DNA repair; (ii) in 464 PhrB homologs, this residue is either Tyr or Phe (Fig. S1†). However, experimental mutation of Tyr391 to Ala blocks FAD photoreduction and DNA repair, underlining its relevance in electron transfer process. This observation rules out other alternative pathways e.g. through Tyr395 or water molecules.
In both WT and Y391F mutant we find an energy barrier at site A. Therefore, we considered a super-exchange tunnelling process for both residues, which is a viable hypothesis to explain the experimental charge transfer between FAD and Trp390 in Y391F mutant where a large energy barrier occurs, but the computed couplings are sufficient to allow charge transfer according to this mechanism.70 We observe similar electronic couplings between FAD and B when a Tyr or Phe aromatic cycle is included as a bridge. One can notice that in WT and Y391F, the Tyr391 or Phe391 aromatic cycle is parallel to the isoalloxazine ring of FAD and in a suitable conformation for π–π orbitals interaction.
Hole transfer via tyrosines is usually not favourable due to the high ionisation potential, which is 0.6 eV larger than for tryptophans (Table 2). The oxidised state of tyrosines can be stabilized by proton transfer, but there is no proton acceptor in the neighbourhood of Tyr391. Therefore, an efficient hopping mechanism, where Tyr391 is transiently oxidized, seems improbable at first sight. The energy of the Tyr391 HOMO, however, is significantly reduced in PhrB. The Tyr391 oxidized state is stabilized by the protein environment instead, in the immediate environment a hydrogen-bond network (Fig. 6) can attract the proton from Tyr391 and allow the O–H bond elongation to compensate an electronic density decrease on the cycle. Therefore, the charge transfer from FAD to Bvia a transiently oxidized tyrosine seems to be possible. In addition to tunnelling, a much more efficient hopping regime seems to be enabled in PhrB due to the specific protein environment of site A. Mutation of Tyr391 to Phe disables this efficient pathway, but does not impede tunnelling as shown by both, experimental and theoretical results. Mutation to Ala, however, blocks the pathway, as shown in experiments and calculations, and thereby blocks FAD photoreduction due to vanishing electronic couplings.
Most photolyases and cryptochromes carry a Trp triad. In these cases, the strong exothermicity of the charge transfer results from stabilization of the positive charge by the solvent, as discussed in detail in our previous work.22–24 The question arises therefore, why PhrB makes use of a Tyr at site A instead of a Trp residue.
Electron transfer can be described by an energy landscape as shown schematically in Fig. 11. It is clear that a Tyr substitution would introduce significant barriers into the charge transfer pathway when it would be placed into the photoreduction pathway of e.g. E. coli photolyase. The barrier, when estimated using the gas-phase ionisation potentials (Table 2) is in the order of 0.6 eV, which surely would block an efficient hopping type charge transfer, as found for proteins with a Trp-triad. In PhrB, however, both Tyr and Trp at site A show a stronger stabilization, due to interactions with water and the protein environment, when compared to other members of the cryptochrome–photolyase family which we have already simulated.23,24 Since site A in PhrB is very close to water molecules in the binding pocket, the intrinsic IP of Tyr and Trp are substantially stabilized by this polar environment. This stabilization results in a downhill hole transfer from FAD to C in PhrB WT but in similar energies for site A and B in Y391W mutant. The calculations suggest a reason for the presence of Tyr instead of Trp in PhrB. The forward transfer is less efficient for Tyr compared to Trp due to the slightly higher IP, however, the back-transfer seems to be too efficient in Y391W mutant, which may lead to unproductive cycles. The Y391W mutation, because of the higher intrinsic IP of Trp, disrupts the downhill energetics and allows charge recombination of FAD. The protein may therefore trade a slightly less efficient forward transfer for blocking back-transfer when using a tyrosine at site A.
Fig. 11 Relative site energies (from left to right) of FAD, site A, site B and site C. For site A, three residue types have been investigated, Trp, Tyr and Phe.
Absorbance spectra showed that the Y391W mutant has lost both FAD and the antenna chromophore DMRL almost completely. The loss of FAD could be due to a modification of the chromophore pocket by Trp, which is larger than Tyr. The small percentage of DNA repair by the Y391W mutant suggests that a small fraction is still able to bind FAD. Quantitative comparisons between photoreduction of Y391W and WT are not possible experimentally. However, our simulations of the Y391W mutant (where loss of FAD was not considered) provide valuable insight of the importance of the Tyr residue in comparison with previously studied cryptochromes and photolyases.23,24 The radical pair state of RP-A is stabilized in our Y391W simulations due to strong electronic couplings and small energy gaps between Trp390 and Trp391 and charge is transferred to Trp342 within 33–70 ps (Fig. 10). Although rate constants for the hopping mechanisms in the Y391W mutant of PhrB are comparable with the Trp triads in E. coli photolyase and Arabidopsis cryptochrome,23,24 backward charge transfers to A occurs more frequently in the PhrB mutant than in these two proteins. Such back transfer increases the risk of charge recombination on FAD and hence a more likely inefficient charge transfer mechanism. If a Trp residue corresponded to the first site and if the FAD binding were efficient, the environment would stabilize the RP-A state in PhrB obviously more than in Trp triads of other members of the cryptochrome–photolyase family.
Taken together, our experimental and theoretical results indicate the following: the protein environment is quite different in PhrB from other groups of photolyases and cryptochromes. Residues bigger than Tyr at position A result in loss of FAD binding. Solvent can come closer to A, stabilizing the FAD˙−–A˙+ state due to reorganization energy. The presence of a Tyr residue instead of a Trp at this site preserves the structure, the energetics, and therefore the function of PhrB.
Computational resources were provided by the state of Baden-Württemberg through bwHPC. Natacha Gillet thanks the Alexander von Humboldt Foundation for a postdoctoral research fellowship.
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