Primary Structure of Tyrosinase fiom Neurospora crassa” 11. COMPLETE AMINO ACID SEQUENCE AND CHEMICAL STRUCTURE OF A TRIPEPTIDE CONTAINING AN UNUSUAL THIOETHER

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methioninecontaining overlap peptides were isolated. “he required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CBl-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 36S, [2,5-3H]histidine, and [5-3Hl histidine-labeled Neurospora tyrosinase.

Portions of this paper (including "Experimental Procedures," Figs. 5-15, and Tables 111-XI1 are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 81M-2727, cite authors, and include a check for $7.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. 50 (Fig. 5) and subsequent ion exchange chromatography of the medium-size molecular weight fraction (pool 2 of Fig. 5) on SE-Sephadex (Fig. 6) allowed the isolation of three fragments (T1 to T3), two of them containing methionine (Table  111). Peptide T1 was subjected to automatic Edman degradation (Table V) and its NH2 terminus was found to be identical with that of the COOH-terminal tryptic peptide of the cyanogen bromide fragment CB3. However, since only the fist eight residues of T1 were determined, the fragment was further digested with thermolysin and the peptide mixture separated by ion exchange chromatography on Beckman "72 ( Fig. 7). The amino acid sequence of the methionine-containing pentapeptide T I -T h l (Table 111) was elucidated by aminopeptidase M and carboxypeptidase C digestion as Ile-His-Gly-Met-Pro (Table IV). This peptide established the order CB3 3 CB1, since only fragment CB1 has an NH2-terminal proline residue.
Automated sequence analysis of the tryptic fragment T2 containing two methionine residues allowed the determination of the fist 15 residues (Table V). The NHZ-terminal sequence (residues 1-7) was found to be identical with the sequence of the COOH-terminal tryptic peptide from CB1, whereas residues 8-15 coincided with the NHz-terminal sequence of CB4. These results unequivocally established the order CB1 "* CB4. The tryptic peptide T3, although devoid of methionine, was also subjected to automatic Edman degradation (Table  VI) and found to be a valuable overlap fragment of three tryptic peptides from CB2 (ct preceding paper, Ref. 1). The alignment of the four cyanogen bromide fragments was further corroborated by the isolation of methionine containing peptic peptides (Fig. 1). Ion exchange chromatography on Beckman "72 ( Fig. 9) and Sephadex G-25 chromatography (Fig. 10) of a peptic digest of in vivo "%-labeled Neurospora tyrosinase resulted in four radioactively labeled peptides (P1 to P4), three of them containing methionine (Table VIII). Compositional and partial sequence analysis (Table IX) of the peptic peptides P1, P2, and P4 were in complete agreement with the alignment of the CB peptides in the order CB3 + CBl -+ CB4 + CB2. This order is further supported by automated sequence analysis of the tryptic fragment T m l (Table VII) isolated from maleylated tyrosinase (Fig. 8, Table 111) and an overlap cyanogen bromide fragment, CB2-4, (Fig. 1, Table X) whose isolation was described in the preceding paper (1).
Chemical Structure of the Tripeptide (Residues 94-96) Containing a n Unusual Thioether-As was pointed out in the preceeding paper (1) residues 26 and 28 of the cyanogen bromide fragment CB1 (residues 94 and 96 in Fig. 2) could not be unambiguously identified. Although back hydrolysis of PTH derivative 26 had suggested a serine residue, this assignment was not compatible with the amino acid composition of the corresponding peptic peptide (residues 91-96). However, compositional analysis of this peptide revealed, in addition to

P Y S T F V A Q E G E S Q S K S T P L E P F W D K S A A N F
Complete amino acid sequence of Neurospora tyrosinase. Cysteinyl residue 94 is covalently linked to histidyl residue 96 via a thioether bond. For the one-letter notation for amino acid residues, see J. Biol Fig. 9. In addition to the three methionine-containing peptides P1, P2, and P4 already described, a fourth fragment P 3 was isolated with the same amino acid composition (Table VIII, Fig. 14) as the peptic peptide from CBI mentioned above (residues 91-96). The specific radioactivity of the 35S-labeled fragment was identical with that of the three methionine-containing peptides (Table I), thus strongly indicating the presence of a modified sulfur-containing amino acid in this peptide. The occurrence of histidine in the same peptide P3 is inferred from the elution profile of a peptic digest of in vivo labeled [2,5-3H]histidine tyrosinase (Fig. 11). In addition to four well resolved radioactive peaks accounting Thr.
for the total of nine histidyl residues in the amino acid sequence of Neurospora tyrosinase (Fig. 2), the profile revealed a fifth component eluting in the same position as P3.
The specific radioactivity of this fragment was found to be only half of those of the other histidine-containing peptides (Table I) indicating a tritium loss in either the 2 or 5 position of the imidazole nucleus. To determine the position from which tritium was lost, tyrosinase labeled in vivo with [5-3H] histidine was again cleaved with pepsin. The elution profile ( Fig. 12) was indistinguishable from the one of Fig. 11 with the exception that the specific radioactivity of P3 was now identical with that of the other histidine-containing peptides ( Table I). The results of the radiolabeling experiments are compatible with a covalent linkage between a cysteinyl residue and C2 of a histidyl residue presumably via a thioether bridge.
In the presence of 6 N HCl a t 110 "C such a structure would be expected to undergo facile cleavage to yield thiolhistidine (SH-His) as one of the products. This conjecture is strongly supported by the observation that synthetic 2-thiolhistidine coelutes with the unknown species of the acid hydrolysate of P3 from cationic exchange resins (Fig. 14). Thus, the linkage of the sulfur in position 2 of the imidazole nucleus is borne out both by the radiolabeling data and by amino acid analysis.
T o c o n f i i further the presence of a thioether structure in P3, the 2,5-3H-labeled fragment was cleaved with chymotrypsin yielding two peptides: the tripeptide P3-C2 (Gly 91-Gly 92-Tyr 93) and the radioactively labeled peptide P3-Cl giving rise to Thr and SH-His after acid hydrolysis (Fig. 13, Table  XI). Treatment of the acid hydrolysate of P3-C1 with a 10fold excess of hydrogen peroxide led to the formation of free histidine and threonine, again supporting the presence of His in this fragment. In addition, peptide P3-Cl gave a positive chloroplatinic acid test (7) and yielded cysteic acid after performic acid oxidation (5) of a sample treated with AgzS04, a reagent known to cleave thioethers (6). These data are thus compatible with the presence of an intramolecular thioether bridge between a cysteinyl and a histidyl residue in this fragment. The complete chemical structure of P3-C1 was established by manual Edman degradation ( Table XII). The first step yielded Ala after back hydrolysis of the ethyl acetate phase, strongly indicating the presence of a cysteinyl residue in position 1. The second amino acid residue was unambiguously identified as Thr in agreement with the results from automatic Edman degradation of CB1 (1). Direct amino acid analysis of the residual aqueous phase after step 2 gave SH-His in good yield, thus codinning the sequence position of the histidyl residue. The manual Edman degradation of P3-C1 was carried out both with the [2,5-3H]histidine and 35Slabeled peptide. In both instances, the bulk of the radioactivity was confined to SH-His (Table XII).
The presence of a thioether structure between Cys 94 and positon 2 of His 96 is also supported by the characteristic absorption features of P3-Cl in the UV region. The absorption spectrum (Fig. 3) was found to be strikingly similar to 2thiolhistidine. The strong absorption of P3-C1 ( E~~~~ = 14,400) is also expected to be manifested in the absorption spectrum of Neurospora tyrosinase (Fig. 15). Indeed, the ratio of A280,260 was found to be rather low (1.50). The calculated ratio based on the content of aromatic amino acids gave a value of 1.71 for Neurospora tyrosinase. The discrepancy between these two values is accounted for by the presence of the strongly absorbing thioether in Neurospora tyrosinase. The complete chemical structure of the tripeptide P3-C1 is depicted in Fig.  4 with its pertinent characteristics summarized in Table 11.
Amino Acid Sequence of Neurospora Tyrosinase-The complete primary structure of Neurospora tyrosinase as deduced from the sequence of the cyanogen bromide fragments (1) and the methionine-containing overlap peptides (Fig. l), is shown in Fig. 2. The enzyme consists of 407 amino acids with a molecular weight of 46,000 including two g-atoms of copper. This value is substantially higher than those reported earlier (8,9); however, the amino acid composition calculated from the amino acid sequence was found to be in good agreement with the published results by Fling et at. (8) if corrected for the larger molecular weight of 46,000.  (residues 94-96, Fig. 2) of Neurosporn tyrosinase. The sulfur of cysteinyl residue 94 is covalently l i e d to the imidazole nucleus of histidyl residue 96 i n position 2. Post-translational Modifications-It was recognized very early in the course of the sequence determination of Neurospora tyrosinase that the NH2-terminal residue is blocked. Using mass spectrometry (IO), the fist amino acid was identified as N-acetylserine. T h i s is in contrast to the four isozymes of mushroom tyrosinase (11) which were reported to contain a free NH2-terminal isoleucine residue. However, mushroom tyrosinase was recently found to be composed of two subunits (12) and therefore it cannot be ruled out that one of the two subunits is also blocked.
In agreement with earlier findings (8) freshly isolated, denatured Neurospora tyrosinase contains no free sulfhydryl group. Hence, the thiol side chain of the sole cysteinyl residue 94 found in this enzyme is quantitatively linked to histidyl residue 96. Although the possibility of an isolation artifact has to be considered, it seems much more likely that this unusual cross-link is an inherent structural feature of Neurospora tyrosinase, generated in a post-translational event. That some processing does occur is suggested by the findings of Fox and Burnett (13) who showed that in crude extracts of the fungus the enzyme is present in an active and an inactive form (protyrosinase). The results of a kinetic study of the activation process of protyrosinase were interpreted in terms of an enzyme-catalyzed intramolecular rearrangement of the molecule. Thus, it is conceivable that the activation of protyrosinase takes place by the enzymatic formation of the thioether linkage between Cys 94 and His 96. In this context it is of interest, that Neurospora crassa is capable of synthesizing ergothioneine (X), an N-trimethylated derivative of 2-thiolhistidine. Biosynthetic studies have indicated that ergothioneine is derived from histidine and that cysteine is the principal source of the sulfur of ergothioneine (15). Thus, possibly related enzymic processes might be responsible both for parts of the synthesis of ergothioneine and for the generation of the thioether structure in Neurospora tyrosinase.
Finally it was of interest to see if this peculiar thioether structure is a unique feature of Neurospora tyrosinase or if it occurs also in other copper proteins. By taking advantage of the ease in converting 2-thiolhistidine into histidine with hydrogen peroxide, a diagonal procedure was developed (see "Materials and Methods"). With this method, no histidine was detected in the hydrogen peroxide-treated amino acid eluate of tyrosinase from the procaryote Streptomyces glaucescens (3). The thioether structure was also absent in Neurospora laccase (2) and Cancer pagures hemocyanin, two copper proteins containing a similar binuclear copper site to Neurospora tyrosinase.
Sequence Homology with Other Copper Proteins-Neurospora tyrosinase is a monoxygenase containing a binuclear copper active site (16)(17)(18)(19)(20). This copper complex, referred to as type 3 copper (21), is shared by the oxygen transporting hemocyanins (22) and by the multicopper oxidases laccase, ascorbate oxidase, and ceruloplasmin. With the exception of ceruloplasmin, however, only limited sequence information is available on these copper proteins. In a recent report by Dwulet and Putnam (23) on the primary structure of the 50,000-and 19,000-dalton fragments of ceruloplasmin, a distinct sequence homology in the active-site residues was observed to the blue copper proteins plastocyanin and azurin as well as to copper-zinc superoxide dismutase. Upon comparison of the COOH-terminal part of the 50,000-dalton fragment of ceruloplasmin with the sequence of Neurospora tyrosinase, a common Leu-His-His sequence (255-257 in ceruloplasmin and 304-306 in tyrosinase) was recognized. This admittedly quite limited sequence homology is of interest since the second of the juxtaposed histidyl residues (residue 306) of Neurospora tyrosinase has recently been demonstrated to be selectively destroyed during active-site directed inactivation by catechol (24). Since, concurrently with the inactivation, one of the two copper atoms was lost, it was suggested that histidyl residue 306 functions as a copper ligand.