lead 
 poisoning is one of the most serious environmental health 
 hazards , with a particularly acute effect on young children . the u.s .
centers for disease control and prevention 
 estimated that approximately 2.5% of children aged 15 years 
 in the u.s . have elevated blood lead levels .
the percentage of affected children in other countries is expected 
 to be even higher .
lead poisoning causes learning disabilities , behavioral 
 problems , and , at very high levels , seizures , coma , and even death .
the 
 mechanism of pb toxification mainly involves binding 
 of pb to proteins and the subsequent inhibition of the 
 proteins physiological functions in blood and tissues .
the documented proteins targeted by pb include several zinc enzymes or proteins ( such as -aminolevulinic 
 acid dehydratase ( alad ) , acetylcoline esterase , cys2his2  zinc - finger  proteins , and acid phophatases ) and calcium - binding proteins ( calmodulin , calbindin , 
 and troponin c ) .
pb can replace 
 zinc and calcium at the oxygen / nitrogen / sulfur - rich active sites of 
 these proteins , thereby inhibiting the protein functions by altering 
 their coordination chemistry and native structures .
for example , the 
 function of alad , an enzyme involved in the second step of heme biosynthesis , 
 is altered by pb binding at the active site via a trigonal 
 pyramidal geometry .
consequently , the hemoglobin synthesis is blocked , 
 leading to anemia . to reduce the pb - induced toxicity ,
organisms have developed various defensive 
 mechanisms with species such as metallothioneins ( mts ) , glutathione , 
 phytochelatins , and lead - binding proteins ( pbbps , which are non - mt 
 acidic proteins that have not been fully characterized ) .
mts , a class of thiol - rich ( up to 30% of its 
 amino acid residues ) , low - molecular - weight proteins whose abundance 
 is particularly high in the liver and kidneys of mammals , are perhaps the most important species for lead 
 detoxification .
mts is crucial to a better understanding of the chemical 
 stabilities , biological functions , and detoxification mechanism of 
 pb  mts .
however , due to the absence of single crystals of pb  mts , 
 information about the pb 
the coordination and binding stoichiometry between mts and metals 
 are dependent on the type of metal ions .
usually , divalent metal ions , such as cd and zn , are tetrahedrally coordinated by four cysteine sulfurs 
 and bind to mts with a stoichiometry of 7:1 .
it can be complexed by a combination of s , o , n , and p - donor ligands 
 with a coordination number ranging from 2 to 9 .
when pb binds to sulfur - rich proteins , three 
 sulfurs in a trigonal pyramidal geometry and the pb 6s lone - pair electrons occupying the axial position ( hemidirected ) 
 constitute the coordination sphere . a 
 number of complexes containing the pb 
s3 coordination 
 have been observed using spectroscopic and mass spectrometry ( ms ) 
 methods . in the presence of biomolecules 
 possessing several distinct donor ligands (
n , o , and s ) , the pb coordination chemistry is diverse and includes formation of pbsxoy and pbsxny .
owing to the presence of o- and s - donor 
 ligands in mts and the unique electronic configuration of pb , coordination of pb with mt is of higher complexity 
 than that of zn or cd .
visible ( uv  vis ) spectra and microcalorimetry 
 have suggested that different pb 
palacios 
 et al . demonstrated with electrospray ionization mass spectrometry 
 that the metal content in pb 
mt2 complexes is dependent on 
 the solution ph ( neutral or 4.5 ) . using 
 extended x - ray absorption fine structure ( exafs ) , vasak et al .
however , crucial questions such 
 as the difference of pb coordination and the chemical 
 stability and physiological function of the two pb 
theoretical 
 calculations have been widely 
 used for the prediction of structures and spectra of metalloproteins .
the combination of quantum mechanics and molecular 
 mechanics - based hybrid ( qm / mm ) method allows two or more computational 
 methods to be performed in a single calculation , making it possible 
 to investigate the chemistry of complex systems with high precision . in this context , the oniom ( our own n - layered 
 integrated molecular orbital + molecular mechanics ) scheme is a general 
 approach because it can combine any number of molecular orbital and 
 molecular mechanics methods .
the oniom 
 method has been successfully utilized in the elucidations of structural 
 and functional properties of many metalloproteins such as cyt c and 
 azurin . in our study ,
optical methods 
 ( uv  vis absorption and circular dichroism ( cd ) spectrometry ) 
 and nmr were used in tandem with the two - layer oniom method to investigate 
 the effect of ph on the structures of two different pb 
the differences in the coordination chemistry of the metal 
 centers and the protein structures at various ph were deciphered . 

we also investigated the chemical stabilities and structural flexibility 
 of these two complexes in proteolytic processing to gain insight into 
 the lead detoxification process involving mts .
zn - containing 
 mt2 , isolated from rabbit liver , was purchased from hunan lugu biotechnology 
 co. ( changsha , china ) .
individual metal - free domains ( apo-mt2 
 and apo-mt2 ) of both rabbit liver and human mt2s , and the corresponding 
 mutants ( d25n - apo-mt2 and d2n - apo-mt2 ) were synthesized 
 by shanghai apeptide co. ( shanghai , china ) . all the domains and their 
 mutations
were confirmed by mass spectrometry performed by the vendor , 
 and the corresponding purity values ( > 95% ) were determined with 
 hplc - ms .
pb ( 99.1% ) was acquired from isoflex usa ( san 
 francisco , 
 ca ) .
lead nitrate , 5,5-dithio - bis-(2-nitrobenzoate ) ( dtnb ) , 
 and cathepsin b were purchased from sigma - aldrich ( st .
deionized water with resistivity 
 of 18.2 m cm was collected from a millipore simplicity 185 
 system ( millipore co. , billerica , ma ) .
all solutions were prepared 
 with deionized water and degassed with nitrogen for at least 30 min .
a 3 kda cutoff 
 millipore ( ym-3 ) membrane ( millipore , billerica , ma ) , equilibrated 
 with 0.01 mol l hcl , was used to separate zn from zn7mt2 . after spinning at 13  000 
 rpm for 30 min at room temperature in an eppendorf 5417r centrifuge 
 ( eppendorf , hamburg , germany )
complete removal of zn from zn7mt2 
 was confirmed by the disappearance of the characteristic absorption 
 of zn7mt2 at 220 nm .
the apo - mt2 concentration was determined by assaying thiol groups 
 with ellman s reagent , dtnb .
mt2 
 solution was stored in a nitrogen - saturated flask to avoid thiol oxidation 
 in apo 
vis absorption experiments 
 were carried out on a uv-2450 
 spectrophotometer ( shimadzu , japan ) in quartz cuvettes ( 1 cm path 
 lengths ) .
cd data were obtained with a jasco-810 spectrophotometer 
 ( jasco corporation , japan ) .
zeta 
 potentials of individual apo - mt2 domains or their mutants were measured 
 at room temperature in a folded capillary cell with a zetasizer nano 
 zs instrument ( malvern instruments , southborough , uk ) .
pb was dissolved 
 with 0.15 m trace metal grade nitric 
 acid ( fisher scientific ) at 250 c , and the pb(no3)2 precipitate was collected , dried , and weighed . 

appropriate amounts of mt2 ( 250 m ) , kcl ( 10 mm ) , and d2o were mixed under n2 atmosphere .
both pb7mt2 complexes were prepared by adding pb(no3)2 to obtain an mt2/pb stoichiometry 
 of 1:7 at corresponding ph ( 7.0 or 4.0 ) .
the resultant solutions were 
 allowed to incubate for 1 h , and the solution ph was brought up to 
 ph 7.0 with koh .
d2o was added to a final volume of 1 ml , 
 and the solution was transferred to an nmr tube .
all pb nmr spectra were recorded on a bruker advance drx-400 mhz spectrometer 
 at 25 c using 60 pulses , a 2 s relaxation delay , and a 
 0.12 s acquisition time ( spectral width of 555.6 khz ) .
a linear prediction 
 was performed to remove the noise , and the real free induction decay 
 ( fid ) was determined before data processing .
after zero - filling , the 
 data ( 128  000 data points ) were processed with an exponential 
 line broadening of 5 hz using the software topspin nmr .
the pb nmr chemical shifts are reported downfield from tetramethyllead 
 (  = 0 ppm ; toluene ) using 1.0 m pb(no3)2 salt ( fisher ) as an external standard (  = 2990 ppm , 
 d2o , 25 c ; relative to tetramethyllead ) . both pb7mt2(i ) and pb7mt2(ii )
complexes were freshly prepared and diluted in 10 
 mm kcl solution ( ph 7.0 ) at 37 c to desired concentrations . 

cathepsin b ( 1.8 ng ) , with a specific activity of 3000 pmol min g , was mixed with 504 
 pmol of pb7mt2(i ) or pb7mt2(ii ) 
 ( the final concentration of pb was 57 m ) in 10 
 mm kcl solution ( ph 5.0 ) .
oniom calculations 
 were performed to predict the optimal geometries and electronic absorption 
 spectra of pb7mt2 complexes .
the initial atomic coordinates of pb4mt2 
 and pb3mt2 were taken from the corresponding 
 pb  substituted cd4mt2 
 and cd3mt2 , respectively .
the structures 
 of cd4mt2 and cd3mt2 
 were retrieved from the rcsb protein databank ( pdb i d : 1 mrb for the 
 -domain and 2 mrb for the -domain ) , in which the absent 
 hydrogen atoms were added using gaussview 4.0
. the protonation states 
 of titratable residues ( e.g. , aspartic acid and lysine ) of pb4mt2 and pb3mt2 at 
 ph 4.0 and 7.0 were determined using pka values estimated with propka 2.0 .
the electronic structures of pb4mt2 and 
 pb3mt2 were modeled with the inclusion of 
 the protein environment , using the two - layer oniom ( qm / mm ) model . 

the qm region comprises the active sites of pb4mt2 
 and pb3mt2 , and the mm region contains the 
 protein environment ( cf .
spin unrestricted density functional theory ( dft ) with 
 the becke s three - parameter hybrid exchange functional and 
 the lee  yang 
parr correlation functional ( b3lyp ) or 
 pure functional bp86 were used for the qm system , and the dunning 
 basis set aug - cc - pvtz - pp was used for pb and 6 - 31+g * basis set for 
 other atoms for the qm calculations . for 
 the mm region , the protein molecule was treated using the universal 
 force field ( uff ) .
an electronic embedding 
 scheme was adopted to deal with the electrostatic interactions between 
 the qm and mm regions in the qm / mm calculations . on the basis of the optimized geometries ,
vertical 
 excitation energies 
 were computed within the oniom scheme by employing the time - dependent 
 density functional theory ( tddft ) .
tddft 
 calculations were carried out with the density functional b3lyp and 
 bp86 . the trizeta basis set aug - cc - pvtz - pp
was used for pb , and 6 - 31+g * * 
 was used for others .
the results were transformed via the swizard 
 program ( version 4.6 ) into each uv spectrum 
 using gaussian functions with half - widths of 3000 cm .
uv  vis absorption 
 and cd spectra were recorded during the titration of rabbit liver 
 apo - mt2 with pb at ph 7.0 ( figure 1 ) . for simplicity , we termed the pb 
mt2(i ) exhibits a characteristic peak centered at 330 
 nm ( figure 1a ) .
the time - resolved absorbance 
 changes ( inset ) indicates that the complexation reaction is fast ( completed 
 in less than 10 min ) . during the titration of apo 
mt2 with 
 pb at neutral ph , some pb(oh)2 ( ksp = 1.4  10 ) precipitate was 
 formed and affected the absorbance value of pb7mt(i ) 
 at 330 nm , as evidenced by the small fluctuation even after 30 min . 

the precipitation can be avoided by lowering the solution ph below 
 6.0 . as shown in the inset of figure s1 in the supporting information ,
the absorbance value of pb7mt(i ) remains stable after the complexation reaction is completed . 
 as indicated by the mt2/pb stoichiometry ( figure 1b ) ,
the maximum binding stoichiometry is 1:7 , consistent 
 with results reported from the substitution experiment of zn7mt2 with pb . the 
 absorption wavelength ( 330 nm ) and
extinction coefficients (  
  3500 m cm ) of pb7mt2(i ) are analogous to values reported for several 
 pbs3 complexes .
the secondary 
 structural variation from apo - mt2 to pb7mt2(i ) 
 is shown in figure 1c .
this 
 is in contrast with the intense cd peaks of cd7mt2 
 and zn7mt2 between 210 and 290 nm . in zn7mt2 ,
the characteristic cd band centering at 244 nm is attributed 
 to the zn(sr)4 chromophore .
similarly , the cd bands of 
 cd7mt2 at 242 and 262 nm can be attributed to the 
 excitation coupling between adjacent pairs of the cd(sr)4 chromophore .
since the m(sr)4 chromophore leads to the appearance of these cd peaks , the absence of any obvious cd peaks in figure 1c suggests that pb7mt2(i ) adopts 
 a different metal coordination geometry from those of cd7mt2 and zn7mt2 , as alluded to in the introduction . 

the individual - and -domains display uv  vis 
 and cd spectral features ( data not shown ) similar to those of apo - mt2 
 during the titration with pb , indicating that the metal 
 centers in the two different domains have similar coordination spheres
. 
 quantitative analysis confirms that the pb / mt2 stoichiometric 
 ratios are 4:1 and 3:1 in the - and -domain , respectively .
time - dependent 
 ( a ) uv  vis absorption and ( c ) cd spectra 
 in 10 mm kcl solution ( ph 7.0 ) containing 7.20 m rabbit liver 
 apo - mt2 and 20 mol equiv of pb .
( b ) dependence of uv absorption peak 
 at 330 nm upon addition of pb to apo - mt2 .
the 
 pb7mt2(ii ) complex displays dramatically different 
 uv  vis 
 absorption and cd spectra ( figure 2 ) from those 
 of pb7mt2(i ) . in the uv  vis absorption spectra , 
 two intense peaks at 325 and 375 nm appear , with the latter having 
 a shoulder peak at 400 nm . in the cd spectra ( figure 2c ) , a strong envelope with maxima at 240 ( + ) , 265 ( + ) ,
320 
 (  ) , 350 ( + ) , 370 ( + ) , and 395 (  ) is produced isodichroically 
 ( 280 , 340 , and 375 nm ) .
positions of all cd peaks are invariant with 
 the pb / apo  mt2 ratio ( increased stepwise from 
 1:1 to 7:1 ) , but the peak intensity increases with the ratio .
the 
 cd spectra of cd7mt2 have two bands at 240 and 
 260 nm split from the 250 nm band , which corresponds to the conversion 
 of isolated cd(sr)4 to the ( sr)3cd  sr 
thus , the unsplittable 
 pb7mt2(ii ) cd peak is indicative of the absence 
 of excitation coupling between adjacent chromophores .
the multiple 
 peaks in the uv  vis absorption and cd spectra suggest that 
 pb in the pb7mt2(ii ) complex has binding 
 modes that are distinctively different from cd in cd7mt2 .
similar to pb7mt2(i ) , the 
 pb / mt2 stoichiometric ratios in the pb7mt2(ii ) 
 complex are 4:1 and 3:1 in the - and -domains , respectively . 

the uv  vis absorption peaks of pb4mt2(ii ) 
 are slightly shifted , with higher intensity than those of pb3mt2(ii ) ( figure 3d ) .
these 
 differences suggest that the metal centers in the two domains have 
 different coordination geometries . to pinpoint the ligands responsible 
 for coordination of pb in the two different domains at 
 neutral and acidic ph
time - dependent ( a ) uv  vis absorption and ( c ) cd spectra 
 in 10 mm kcl solution ( ph 4.5 ) containing apo - mt2 ( 7.20 m ) 
 and 20 mol equiv of pb .
( inset ) the time - resolved absorbance 
 changes at 325 and 375 nm , respectively .
( b ) the dependence of uv 
 absorption peaks at 325 and 375 nm on the addition of pb to apo - mt2 .
structures and electronic 
 absorption spectra of the - and 
 -domains in ( a , c ) pb7mt2(i ) and ( b , d ) 
 pb7mt2(ii ) obtained by experimental and oniom methods . 
 in panels a and b , the dark gray spheres are pb , yellow 
 spheres are s , red spheres are o , gray spheres are c , and white spheres 
 are h atoms
. the oniom method has been successfully used 
 to predict metalloprotein structures . to validate the method for the studies of mt2 structures , structural 
 optimization of cd4mt2 and cd3mt2 models
compared to the nmr 
 results , the computed structures of the 
 metal clusters display little deviation ( e.g. , the bond lengths have 
 a root - mean - square deviation of only 0.040.05  when 
 compared to the experimental data ) ( figure s2 and table s1 in supporting information ) .
therefore , we conclude 
 that the oniom method is viable for the studies of the two pb7-mt2 complexes .
the theoretical calculations were performed 
 separately on the - and -domains on the basis that the 
 two domains are structurally independent .
the initial atomic coordinates 
 of each domain in pb7mt2 were respectively adopted 
 from the pb - substituted cd4mt2 
 and cd3mt2 because a well - established model 
 of apo - mt2 is not available .
the protonation states of titratable 
 residues ( e.g. , asp and lys ) at different ph were determined from 
 the pka values estimated with propka 2.0 .
in the two - layer oniom model of pb7mt2(i ) , the 
 qm region consists of four pb atoms , and the side chains consist of 
 eleven cysteine residues in the -domain . as for the -domain , 

the three pb atoms and the side chains of nine cysteine residues constitute 
 the qm region .
figure 3a displays the optimized molecular geometries of 
 the - and -domains in pb7mt2(i ) , 
 which are quite different from the well - characterized cd7mt2 and zn7mt2 structures . in cd7mt2 and zn7mt2
, each metal adopts the 
 tetrahedral coordination with terminal and bridging thiolates to form 
 a metal  ligand six - membered ring in the -domain and 
 two fused six - membered rings in the -domain.(cf .
figure s2 
 in supporting information ) however , all 
 seven pb ions in pb7mt2(i ) are trigonally 
 coordinated by three cysteine sulfurs ( pb  s3 ) without 
 any metal 
these 
 significant differences can be attributed to the pb 6s lone - pair electrons , which disrupt the tetrahedral coordination 
 by occupying the axial position with a significant stereochemical 
 activity .
this point is in line with the report by godwin and co - workers , 
 who stated that pb 
s4 is not a preferred coordination 
 and that trigonal pyramidal geometry in all - sulfur coordination is 
 predominant .
moreover , due to the pb  s3 coordination , only one bridging cysteine ( pb6s18pb7 
 in the -domain ) remains , preventing a pb 
according to the corresponding structural parameters 
 ( table 1 ) , the average length of the pb 
s 
 bonds is 2.68  , close to the reported exafs value ( 2.65  ) . the electronic absorption spectra ( red line 
 curves ) , simulated by tddft using the b3lyp functional , are overlaid 
 with the experimental results ( black line curves ) in figure 3c .
the agreement between the simulation and experimental 
 data in the oscillator strengths indicates that the optimized molecular 
 geometries of pb4mt2(i ) and pb3mt2(i ) are reasonable .
detailed analysis of vertical 
 excitation energies , oscillator strengths , and molecular orbital contributions 
 ( table s3 in supporting information ) indicated 
 that four transitions contribute to the absorption peak at 330 nm . 

these transitions are homolumo , homo-1lumo , homo-2lumo , 
 and homo-1lumo+1 . from the frontier molecular orbitals ( figure 
 s3 in supporting information ) , lumo and 
 lumo+1 ( the final state of the transitions )
s bonds , while homo and homo-1 ( the initial states of 
 the transitions ) are largely localized at the sulfur atoms .
therefore , 
 the electronic absorption band at 330 nm can be attributed to the 
 spb ligand - to - metal charge transfer ( lmct ) .
in the two - layer oniom model 
 of pb7mt2(ii ) , 
 we initially assigned the qm region of each domain to be the same 
 as that of pb7mt2(i ) .
however , the optimized molecular 
 geometries revealed two unexpected short pb / o distances ( 5.52  
 for pb1 with the carbonyl oxygen of asp2 in the -domain and 
 4.31  for pb6 with the carbonyl oxygen of asp56 in the -domain ) . 

these short distances should result from the electrostatic interaction 
 between the protein surface ( the mm region ) and the negatively charged 
 pb 
acidic ph conditions , the protein surface is neutral or positively 
 charged , causing the mm and qm regions to move closer , thereby shortening 
 the distance between the pb 
moreover , based on the pka values of asp 
 ( 4.00 ) , both asp residues are neutral in weakly acidic solution , 
 which are more favorable than the negatively charged ( deprontonated ) 
 form to the positioning of the negatively charged pb 
these pb / o distances are close to the sum of the 
 van der waals radius of pb and o ( 3.54  ) , indicating the pbo interaction must be 
 taken into account .
we therefore modified the initial qm region in 
 each domain by including the respective asp residue .
as shown 
 in figure 3b , the optimized molecular 
 geometry of pb7mt2(ii ) shows an entirely different 
 coordination sphere from that of pb7mt2(i ) .
the 
 overall coordination sphere includes the trigonal pyramidal pb  s3 mode , the distorted trigonal pyramidal pb 
s2o1 mode in the -domain , and the distorted quadrilateral 
 pyramidal pb  s3o1 in the -domain . 

the pb 6s lone pair electrons occupy the 
 axial position with a significant stereochemical activity , resulting 
 in hemidirectionality in the pb  ligand coordination .
moreover , due to protonation of the 
 peptide side chain , several cysteine sulfurs ( especially in the -domain ) 
 are closer to the protein exterior and increase the effective radius 
 of the metal center and the pb  s bond length ( cf .
the loosened structure 
 of the metal center renders a higher flexibility to pb7mt2(ii ) .
the simulated oscillator strengths are in good agreement 
 with the experimental data ( figure 3d ) , indicating 
 that the optimized molecular geometries of pb4mt2(ii ) 
 and pb3mt2(ii ) are reasonable . the small 
 deviation between the electronic absorption spectra of the two domains 
 ( cf .
detailed vertical excitation energies and 
 molecular orbital contributions ( table s5 and figure s4 in supporting information ) indicate that all three 
 bands are primarily associated with the spb lmct .
note that all of the above calculations are based on b3lyp , a hybrid 
 functional to fit data primarily for main - group elements .
because of the large exact exchange component 
 ( 20% ) , b3lyp is known to favor loose electron densities and low transition 
 energies . to confirm the reliability of b3lyp , the pure functional 
 bp86 , a generalized - gradient approximation ( gga ) class with zero exact 
 exchange ,
is used as a control study on 
 the geometry optimization and calculations of the excited states for 
 both pb7mt2 complexes .
the simulated structures 
 of both pb7mt2 complexes from bp86 pure functional 
 ( table s69 in supporting information ) are similar to those from b3lyp hybrid functional .
however , the 
 spectra from bp86 pure functional display a significant deviation 
 from the experimental data ( figure s5 in supporting 
 information ) , indicating that the b3lyp hybrid functional is 
 a better choice .
the b3lyp hybrid functional produces better spectral 
 accuracy , which can be attributed to its increased amount of hartree 
 local 
 minima  
 in the qm optimization . in the qm / mm calculation of biomolecules , 
 the vast size of the available configuration space may cause the qm 
 optimization to stop at local minima .
one way to circumvent this problem 
 is to use a reliable structure ( e.g. , a structure deduced from nmr 
 or x - ray crystallography ) at the beginning of the qm optimization .
have used the 
 x - ray structure of azurin as the initial structure in the qm / mm calculation 
 of metal - substituted azurins and obtained noticeable structural changes 
 on the active sites when cu in azurin was substituted 
 by metal ions such as co , ni , or zn . we adopted this approach 
 by using the pb - substituted cd4mt2 
 and cd3mt2 structures deduced from the nmr 
 experiments as the initial structures . 

the theoretical spectra agree well with the experimental results , 
 suggesting a high level of reliability of the calculation and in the 
 predicted structures .
to provide more 
 experimental evidence 
 to our computational results about the two different pb7mt2 complexes , we conducted pb nmr in solutions 
 of the two complexes ( figure 4 ) .
pb7mt2(i ) displays one pb peak at 5679 ppm , while 
 pb7mt2(ii ) exhibit two peaks at 5820 and 4348 ppm . 
 thus it is clear that the pb coordination in these two 
 complexes are different .
furthermore , the pb signal 
 of pb7mt2(i ) at 5679 ppm is well within the chemical 
 shift region ( from 5600 to 5800 ppm ) where pbs3 species 
 with the trigonal pyramidal coordination are observed .
thus we conclude that pbs3 is the binding mode in pb7mt2(ii ) . for pb7mt2(ii ) , the peak 
 at 5820 ppm is also assigned to the pbs3 coordination , 
 given its close vicinity to the 56005800 ppm region .
compared 
 to the pb7mt2(i ) peak , the shift by 141 ppm can 
 be attributed to the changes of the aforementioned pb  s bond 
 length and the s  pb  s bond angle in the pbs3 coordination .
the lower intensity is indicative of the decreased 
 number of pbs3 clusters in pb7mt2(ii ) . 

the conversion of metal centers in the pbs3 coordination 
 to a different binding mode contributes to the appearance of the peak 
 at 4348 ppm .
it is well - known that the nmr signal of pb bound to o - containing ligands is shifted upfield with respect 
 to that bound to s - containing ligands .
pb nmr spectra of ( red ) pb7mt2(i ) , 
 ( blue ) pb7mt2(ii ) , and ( black ) pb(no3)2 at ph 7.0 .
the carbonyl group of asp is the only o - donor ligand in mt2 . 
 to 
 further verify the formation of the pb 
o bond in pb7mt2(ii ) , a titration was performed by adding pb into a peptide solution whose asp residue had been mutated with 
 asn ( i.e. , d25n - apo-mt2 and d2n - apo-mt2 ) .
zeta potential 
 and cd measurements did not show discernible changes in the surface 
 charge and structure of both mutants ( figure s6 and s7 in supporting information ) , confirming the viability 
 of these mutants for coordination studies . at neutral ph ,
both uv  vis 
 absorption and cd spectra of the two mutants show features similar 
 to those in the same spectra of pb4mt2(i ) 
 and pb3mt2(i ) , verifying that asp does not 
 participate in the pb coordination in pb7mt2(i ) . 
 at acidic ph ( ph < 5 ) ,
the uv  vis absorption spectra of 
 both mutants display only a single peak at 330 nm , and the cd spectra 
 did not reveal any changes . while these features are rather different 
 from the corresponding spectra of pb4mt2(ii ) 
 and pb3mt2(ii ) , they are analogous to those 
 of pb4mt2(i ) and pb3mt2(i ) . 
 we therefore conclude that asp is involved in the coordination of 
 pb in pb7mt2(ii ) . the unique uv
 vis 
 absorption spectra of pb7mt2(ii ) , with multiple 
 peaks and the presence of a new pb nmr peak at 4348 
 ppm , are well - correlated with the mutational study . another 
 piece of evidence for the formation of pb  o bond 
 is the transition from a single peak to three peaks in the time - dependent 
 spectra of a mixture of pb / apo - mt2 at ph 4.55.0 
 as well as the appearance of multiple peaks in the cd spectra ( cf . 

however , because 
 of the negative impact of acidity on the formation of the pb  s 
 bond ( cf . the proton - releasing process shown in reaction 1 ) and the rearrangement of the peptide chain , the pb 
therefore , under 
 more acidic conditions ( ph < 4.5 ) , the time - dependent peak transition 
 disappears , and the spectra show features typical of pb7mt2(ii).1 time dependence 
 of ( a ) uv  vis absorbance and ( b ) cd spectra 
 in a kcl solution after the addition of 7 mol equiv of pb to an apo - mt ( 7.20 m ) solution at ph 5.0 .
reaction times 
 from bottom to top : 0 , 1 , 3 , 5 , 7 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , 
 90 , 105 , 120 , 135 , 150 , 175 , and 180 min . to study the acid tolerance of both pb7mt2 complexes , a series of spectrophotometric titrations 
 at different ph was performed .
the corresponding complex was confirmed 
 by the appearance of characteristic uv  vis absorption peaks 
 of pb7mt2(i ) or pb7mt2(ii ) . 

the results show that pb7mt2(i ) is formed above 
 ph 5.0 , whereas pb7mt2(ii ) is produced at more 
 acidic ph .
pb7mt2(i ) can be transformed to pb7mt2(ii ) by adjusting the solution ph to 5.0 or lower . 

however , once pb7mt2(ii ) is formed , it remains 
 stable at neutral ph .
our demetalation experiments ( figure s8 in supporting information ) indicate that ph 2.5 
 is sufficiently low for the complete removal of pb from 
 pb7mt2(i ) , but stripping pb completely 
 of pb7mt2(ii ) requires a ph as low as 2.0 . these 
 results ( summarized schematically in figure 6 )
indicate that pb7mt2(ii ) has a higher tolerance 
 toward an acidic environment than pb7mt2(i ) has . 

moreover , pb7mt2(ii ) remains stable even in the 
 presence of apo - mt at neutral ph ( data not shown ) .
we believe that 
 the greater acid tolerance and higher structural stability of pb7mt2(ii ) results from the pb  o bond .
such a 
 finding has a significant implication to the lead detoxification process 
 in physiological milieu .
the pb - inflicted toxicity stems from its tight 
 binding 
 to a variety of sulfur - rich proteins , such as gata proteins and the 
 steroid receptor dna - binding domains .
mt2 reduces the pb - inflicted toxicity 
 by seizing free pb or sequestering pb from 
 these proteins to recover the native protein function .
higher acid 
 tolerance and greater structural stability render mt2 a greater power 
 in effectively scavenging pb in different environments . 
 as shown in reaction 1 ,
mt2 accompanies the release of h , which 
 in turn increases the acidity in a highly localized region ( e.g. , 
 in cytosol ) .
moreover , it has been reported that elevated pb concentrations induce the stress level of various organisms , and 
 acidity is also correlated with the stress level .
we posit that pb7mt2(ii ) is more effective 
 than pb7mt2(i ) in lead detoxification , given its 
 greater structural stability and acid tolerance .
elucidation of the metabolism of the pb - mt 
 complexes is vital for understanding the lead detoxification by mts . 

some reports have suggested that exogenous mt is processed mainly 
 by the lysosomal protease , and the rate of mt degradation is dependent 
 on the types of metals bound by mts .
four 
 different cathepsins have been identified in lysosomes , and the cysteine 
 protease ( cathepsin b , l , and h ) is the principal protease for mt 
 degradation .
variations in the concentrations of the two pb7mt2 
 complexes in the presence of cathepsin b were measured by uv  vis 
 absorption spectrometry . in figure 7 , the pb7mt2 concentrations were normalized with respect to 
 their initial concentrations . within 120 min , pb7mt2(ii ) 

such a 
 higher degradation rate can be attributed to the more flexible structure 
 of the pb7mt2(ii ) complex .
cathepsin b is an endopeptidase 
 that cleaves internal peptide bonds and favors a large hydrophobic 
 side chain in the substrate protein .
side 
 chains on the amino acids dock into the cathepsin s subsites , 
 whose interaction with the protein substrate is dependent on the flexibility 
 of substrate protein . in mts ,
the existence 
 of bridging cysteine sulfurs compacts the metal center , which dominates 
 the protein folding . in pb7mt2(ii ) , due to the lack of bridging cysteine sulfurs , the 
 metal center is loosened , which improves the flexibility of the protein . 
 moreover , when the pb 
s3o1 become 
 distorted . consequently , pb ions are positioned farther 
 from the cysteine sulfurs , and the pb  s bonds are weakened . 
 both processes facilitate the conformational adjustment of pb7mt2(ii ) .
the significantly improved flexibility of 
 pb7mt2(ii ) facilitates the protein in the  induced - fit  
 model with cathepsin b. as a result , 
 the proteolytic processing of pb7mt2(ii ) is greatly 
 accelerated . in living organisms ,
the acidity in lysosome is about 
 5 , which is sufficiently low to cause the structural conversion from 
 pb7mt2(i ) to pb7mt2(ii ) and 
 to accelerate the degradation of pb  mt2 complexes .
time - dependent proteolytic processing of ( black ) pb7mt2(i ) and ( red ) pb7mt2(ii ) by cathepsin 
 b. the coordination of pb with individual human apo  mt2 
 or apo  mt3 domains at different ph was also studied by uv  vis 
 absorption and cd spectrometry .
mutational studies revealed 
 that the asp residue is also essential for the ph - dependent structural 
 variation . in line with the data observed for the rabbit liver mt2 , 
 the structure - dependent chemical and biological activities of pb7hmt2(ii ) formed between human mt2 and pb have higher acid tolerance , more coordination stability , and faster 
 proteolytic processing than pb7hmt2(i ) ( figure 
 s10 in supporting information ) .
our results 
 suggest that there exists a commonality in the pb coordination 
 chemistry among mammalian mt2s .
in this work , the ph - dependent coordination chemistry between pb and mt2 was systematically studied .
the combination of spectroscopic 
 studies and oniom calculations provided a detailed description of 
 the two different pb7mt structures .
the results 
 and structures were further verified by pb nmr and mutational 
 experiments .
the similar structural , chemical , and biological properties 
 between rabbit liver pb7mt2(ii ) and human pb7mt2(ii ) suggest a commonality in the pb coordination chemistry among mammalian mt2s .
the higher acid tolerance , 
 greater coordination stability , and faster degradation rate of pb7mt2(ii ) have significant implications for the pb detoxification process .
specifically , mt2 reduces the pb - inflicted toxicity by seizing free pb in the 
 cellular milieu or by sequestering pb from pb - inflicted proteins .
the unique properties of pb7mt2(ii ) 
 render mt2 a greater power to effectively scavenge pb in different environments ( e.g. , in a localized acidic cytosol region ) . 

moreover , the greater flexibility of pb7mt2(ii ) , 
 resulting from the absence of bridging cysteine sulfurs , helps to 
 accelerate its processing by lysosomal protease .
the structural conversion 
 from pb7mt2(i ) to pb7mt2(ii ) 
 is likely to occur in the acidic environment of lysosome , facilitating 
 the effective detoxification and metabolism of pb .
 lead 
 is a toxic heavy metal whose detoxification in organisms is 
 mainly carried out by its coordination with some metalloproteins such 
 as metallothioneins ( mts ) . 
 two pb  mt complexes , named as pb7mt2(i ) and pb7mt2(ii ) , form under 
 neutral and weakly acidic conditions , respectively . 
 however , the structures 
 of the two complexes , which are crucial for a better understanding 
 of the detoxification mechanism of pb  mts , have not been clearly 
 elucidated . in this work , 
 coordination of pb2 + with rabbit 
 liver apo  mt2 , as well as with the two individual domains ( apomt2 
 and apomt2 ) at different ph , were studied by combined 
 spectroscopic ( uv  visible , circular dichroism , and nmr ) and 
 computational methods . 
 the results showed that in pb7mt2(i ) 
 the pb2 + coordination is in the trigonal pyramidal pb  
 s3 mode , whereas the pb7mt2(ii ) complex contains 
 mixed trigonal pyramidal pb  
 s3 , distorted trigonal 
 pyramidal pb  
 s2o1 , and distorted quadrilateral 
 pyramidal pb  
 s3o1 modes . 
 the o - donor 
 ligand in pb7mt2(ii ) was identified as the carboxyl 
 groups of the aspartic acid residues at positions 2 and 56 . 
 our studies 
 also revealed that pb7mt2(ii ) has a greater acid 
 tolerance and coordination stability than pb7mt2(i ) , 
 thereby retaining the pb2 + coordination at acidic ph . 
 the 
 higher flexibility of pb7mt2(ii ) renders it more 
 accessible to lysosomal proteolysis than pb7mt2(i ) . 
 
 similar spectral features were observed in the coordination of pb2 + by human apo - mt2 , suggesting a commonality among mammalian 
 mt2s in the pb2 + coordination chemistry . 