Stellacyanin. Studies of the Metal-Binding Site Using X-Ray

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STELLACYANIN Studies of the Metal-binding Site using X-ray Absorption Spectroscopy

J. PEISACH Departments ofMolecular Pharmacology and Molecular Biology, Albert Einstein College ofMedicine, Bronx, New York 10461

L. POWERS AND W. E. BLUMBERG Bell Laboratories, Murray Hill, New Jersey 07974 B. CHANCE Johnson Research Foundation, University ofPennsylvania Medical School, Philadelphia, Pennsylvania 19104 ABSTRACT Stellacyanin is a mucoprotein of molecular weight -20,000 containing one copper atom in a blue or type I site. The metal ion can exist in both the Cu(II) and Cu(I) redox states. The metal binding site in plastocyanin, another blue copper protein, contains one cysteinyl, one methionyl, and two imidazoyl residues (Colman et al. 1978. Nature [Lond.l. 272:319-324.), but an exactly analogous site cannot exist in stellacyanin as it lacks methionine. The copper coordination in stellacyanin has been studied by x-ray edge absorption and extended k-ray absorption fine structure (EXAFS) analysis. A new, very conservative data analysis procedure has been introduced, which suggests that there are two nitrogen atoms in the first coordination shell of the oxidized [Cu(II)] protein and one in the reduced [Cu(I)] protein; these N atoms have normal Cu-N distances: 1.95-2.05 A. In both redox states there are either one or two sulfur atoms coordinating the copper, the exact number being indeterminable from the present data. In the oxidized state the Cu-S distance is intermediate between the short bond found in plastocyanin and those found in near tetragonal copper model compounds. Above -1400C, radiation damage of the protein occurs. At room temperature the oxidized protein is modified in the x-ray beam at a rate of 0.25%/s. INTRODUCTION structure of the Cu metal-binding site in the oxidized protein to have a geometric array of ligands not very Ever since the original observation of their intense color, different from that of the reduced protein. In addition, the blue copper proteins have been studied in order to NMR (6-8), electron spin echo (9,10), and ENDOR understand the structure that gives rise to their unusual (electron-nuclear double resonance) (11) studies demon- physical properties. Some of these properties include strate that the metal binding site contains one or more intense optical absorptions near 600 nm with molar extinc- imidazole nitrogen atoms, while the linear electric field tion coefficients of 3,000-5,000 cm-1, a nuclear hyperfine effect in pulsed EPR (12) shows that the copper binding interaction in the EPR of 3-9 x 10-3cm-', about one- site has a large internal electric field, consistent with an quarter that of copper complexes having near tetragonal accessible excited state charge-transfer interaction. Not all geometry, and oxidation-reduction potentials 100-600 mV of these properties are reproduced by nonblue copper higher than that for the Cu(II)/Cu(I) couple in aqueous proteins, by model copper complexes made up of amino solution (1,2). acids and peptides, or by model compounds of a more Over the past decade, various structural explanations exotic nature (13,14). based on biochemical and spectroscopic evidence have A major breakthrough in our understanding of the blue been given for these properties that are related to the copper proteins comes from the x-ray crystallographic structural and stereochemical makeup of the copper bind- analysis at 2.7-A resolution and subsequent refinement to ing site. For example, the intense blue color is believed to 1.6 A of poplar leaf plastocyanin (15), a 10,500-dalton (D) arise from a ligand-to-metal charge transfer from a cyste- molecule that contains a single copper atom. The extinc- inyl sulfur (3,4). The EPR properties are thought to be due tion coefficient per copper atom for the analogous protein to the nontetragonal and likely near tetrahedral symmetry from Chenopodium album is 4,700 M-'cm-' at 597 nm of the metal site (5), while the high redox potential is (16), while All, the nuclear hyperfine constant in the EPR attributed to the stereochemical constraint that causes the for this protein (16) is 7.0 x 10-3cm-'. The redox potential

BIOPHYS. J. e Biophysical Society * 0006-3495/82/06/277/09 $1.00 277 Volume 38 June 1982 277-285 for bean plastocyanin is 347 mV (17). For the poplar the EXAFS of this damaged material showed that the protein in the oxidized state, the ligands to Cu(JI) consist binding site for Cu(II) now consisted of two nitrogen and of two histidine imidazole nitrogen atoms and a cysteinyl two sulfur atoms. In the reduced, radiation-damaged sulfur. At a distance too far to constitute a real chemical material, the ligands to Cu(I) are likely to be two nitrogen bond (2.9 A), another sulfur atom, from methionine, is atoms and one or two sulfur atoms, 2.31-2.34 A from the found. A similar ligand arrangement is deduced for copper, which is a normal Cu(I)-S bond distance. another low-molecular-weight, blue copper protein, azurin, in which the same atoms are arranged about the metal METHODS atom (18). Thus, many of the structural predictions based on spectroscopic measurements have been borne out. Sample Preparation Others (19), regretfully, have not. Stellacyanin was prepared by the method of Mims and Peisach (9) from In addition to plastocyanin and azurin, a third blue lac acetone powders purchased from Saito and Company (no. 9, Homma- copper protein that has drawn a great deal of attention chi l-Chome, Higashi-Ku, Osaka, Japan). After the last column chroma- tographic step, those fractions with an Aw./A280> 0.14 were combined. since its discovery by Omura (20) is stellacyanin, a muco- The protein was concentrated under pressure (Amicon Corp., Scientific protein isolated from Rhus vernicifera, the Japanese lac Systems Div., Lexington, MA) and equilibrated against 0.05 M KPO4 tree (20,5). This 20,000-D molecule has physical proper- buffer, pH 7. It was sometimes found that our stellacyanin preparations ties that relegate it to the class of blue copper proteins, yet were only slowly reduced with dithionite, asjudged from loss ofblue color. they are decidedly different from those of azurin and We could, however, reduce the protein by using long-term anaerobic plastocyanin. To begin with, the redox potential of stella- incubation with a large excess of dithionite. cyanin, 184 mV, is decidedly lower (17). The EPR spec- Monitoring trum of stellacyanin is indicative of rhombic symmetry To ensure protein integrity and redox state, optical spectroscopy (5,21), whereas those for azurin (22) and plastocyanin was used to monitor the sample before, during, and after exposure to the (16) indicate an axially symmetric site. A more fundamen- x-ray beam. The samples were maintained at - 1400C or lower to prevent tal difference is in the amino acid composition. Stella- damage by hydrated electrons and free radicals produced during exposure cyanin contains no methionine (5,23), which is a conserved (24). However, in the case shown in Fig. 8, a temperature of - 500C was near neighbor of copper in all azurins and plastocyanins. used to demonstrate optical bleaching. In addition, EPR measurements were made before and after each 10 min of x-ray exposure. The One finds a cysteinyl residue in the portion in stellacyanin specifically designed optical spectrophotometer, sample holders, and EPR said to be homologous with these methionine-containing spectrometer used in this study are described elsewhere (25). regions (8). Although stellacyanin would be an attractive candidate for x-ray crystallographic study, the growth of a Measurements to suitable crystal has proven be an intractable problem, All x-ray absorption measurements were made at Stanford Synchrotron probably due to solubility properties which are in part Radiation Laboratory during dedicated operation of the SPEAR storage governed by the large carbohydrate content of the mole- ring at 40-80 mA and -3.0 GeV. Beamline I-5 was used exclusively for cule (5). For this reason, we have turned our attention to both edge and EXAFS measurements providing -1 x 10'0 photons/s. the EXAFS technique for the elucidation of the metal- The initial intensity was monitored by an ionization chamber preceded by a defining slit. The fluorescence was measured using the filter-fast binding site in this protein. plastic scintillator assembly described by Powers et al. (25). The samples, From the analysis of the data, we conclude that the prepared in holders suitable for the cryostat (24,25), were maintained at ligands to the metal in oxidized stellacyanin are most - 1400C or lower at all times. probably two nitrogen atoms and one sulfur atom. The Cu-S bond length was determined to be -2.21 A, consid- Edge Spectra erably larger (by at least 0.05 A) than the analogous bond Edge spectra were analyzed using the methods described previously (26). in plastocyanin and closer in distance to that found in the The error in absolute energy is ±0.1 eV. The EXAFS data (Fig. 1) were near planar CuN2S2 complex, Cu(II)-bis-thiosemicarba- analyzed by the methods described by Lee et al. (27) and Powers (28). zone, (2.26 A). There is no evidence for the presence of a methionyl sulfur analogue >2.5 A away from the copper. EXAFS Spectra There is, however, a ligand that may be sulfur at -2.8 A The modulation of the x-ray absorption spectrum by the EXAFS is given but that could also be nitrogen, carbon, or oxygen, or these by in combination. In the case of reduced stellacyanin, one cannot unambiguously choose the correct mathematical -N, If(k w) 12 (e-22_'-1/) sinl2[kr, + ad(k)]l, solution from the analysis, but a first coordination shell ,kri consisting of one nitrogen and one sulfur atom provides the where the sun is over the distances r, from the absorbing atom, Nf is the best fit to the data. number of the same type of backscattering atoms at r,, X is the During the course of our studies, we found that stella- photoelectron mean free path andfi(k, -r) is the backscattering amplitude of the ith atom which is -Z/k2 (for atomic number Z) when k > -4 A-'. cyanin protein easily became damaged by synchrotron The disorder parameter r2 (sometimes called the Debye-Waller factor) radiation as demonstrated from changes in the EPR and describes the mean-square displacement in r, that arises from thermal optical properties of the oxidized protein. An analysis of and/or lattice disorder. The phase shift a(k) is the energy-dependent

278 BIoPHYSICAL JOURNAL VOLUME 38 1982 a

w 0

-J 0. 4 L 02 a 4 -J LI

- ~ ~ ~ ~ ~ b

1. . I . .-A. I . . . . I . ., . . I . . .. I . I . . I . 0 2 4 6 8 10 9000 9200 9400 9600 r+a (K) X-RAY PHOTON ENERGY (eV) FIGURE 3 Fourier-transformed data for (a) oxidized and (b) reduced FIGURE I X-ray absorption spectra of (a) oxidized and (b) reduced stellacyanin. Note that the abscissa contains the phase a (k). stellacyanin at - 1400C. A linear background that sets the absorption below the edge equal to zero has been removed. Cu(II)-diethyldithiocarbamate (Cudtc), which has four sulfur atoms at an average distance of 2.316 A (30, 31). The fitting program used to phase resulting from the potentials of both the absorbing and backscatter- compare the models with back-transformed data is described by Brown et ing atom, and k is the wave vector given by al. (32). The variable parameters for each of the two atom types, denoted by subscripts N and S, are r, the average distance from the copper atom; k = (2 m,[E - Ej)2/'lh N, the number of atoms at this average distance; Ao?, the change in disorder contribution with respect to the model; and AE., the change in where me is the electron mass, E is the x-ray photon energy, and EO is the edge or threshold energy with respect to that chosen for the model. The edge or threshold energy. Fig. 2 shows the k3x(k) data for both oxidized sum of the residuals squared, ZR?, is useful when comparing different and reduced stellacyanin. These data were Fourier transformed without fits having the same input data. The parameters are not orthogonal; the smoothing or use of a filter function and are shown in Fig. 3. Using a largest correlations are generally found between N and Ac?. Standard Fourier filter, each resolved shell was back-transformed (Fig. 4) and analytical methods were used to determine these correlations and the resolved into an amplitude and a phase function. directions of largest change. Model compounds for Cu-N and Cu-S amplitudes and phases for The fitting algorithm was applied in a novel way that gave us increased first coordination shells were Cu(II)-tetraphenylporphine (CuTPP), insight into the mathematical behavior of this complicated analytical which has four nitrogen atoms at an average distance of 1.984 A (29), and problem. The solution was considered to consist of nitrogen and/or sulfur atoms as copper ligands exclusively. The two parameters related to NN and Ns were held fixed and the other six were permitted to vary so as to find a minimum ER?, a procedure that is always at least locally convergent. For each such mathematical solution, a decision was made as to whether it was physically acceptable or not (criteria are discussed

- 2. x. V in

X2.0 -

-2

-. . I .. -' E . -^*+... * * I S. ;. I * . I I I F. ~"I--J-. a... , . - . -. 4 I ' . ..^_ 0 2.5 5 7.5. 10 12,5 : , 4 6 , a I 10 12. K (A-) K(A-,) FIGURE 2 Background removed or total EXAFS data multiplied by k3 FIGURE 4 First-shell filtered data of oxidized (-) and reduced (+) for (a) oxidized and (b) reduced stellacyanin. stellacyanin.

PEISACH ET AL. Metal-binding Site ofStellacyanin Studied by EXAFS 279 below). Then a locus containing all physically acceptable solutions could used to identify unambiguously the chemical type of be circumscribed about all such points. Then the six variable parameters ligands. were set to widely differing initial values to search for alternative local minima. A well-conditioned data set will have one such minimum (a global one), but arbitrary data may have as many minima as are EXAFS Data permitted by the number of degrees of freedom in the data. Oxidized state. It is complicated to fit the first- RESULTS shell-filtered stellacyanin data to nitrogen and sulfur, the X-ray Absorption Edge Data probable ligands, because the difference in average distance of the nitrogen and sulfur ligands to the copper atom The structural features of the absorption edge contain is too small for each contribution to be resolved in the data information about the molecular energy levels of the (i.e., the first shell peaks in the Fourier transforms are absorbing atom. The systematics of these structural fea- single peaks and the filtered data of these contain no beat tures have been reported elsewhere for copper model nodes). The parameters N and Aa2 are highly correlated compounds and proteins (26). Fig. 5 compares the edges of for each type of ligand atom, and, in addition, the ampli- oxidized and reduced stellacyanin. tudes for nitrogen and sulfur are similar enough so that The oxidized protein displays a Is -3d transition that reasonable changes in Ao3 make them difficult to distin- is expected for most Cu(II) compounds. As discussed guish. For these reasons, the EXAFS technique cannot previously (26, 28, 33), the observation of this transition uniquely discriminate their respective contributions. clearly identifies a Cu(II) or Cu(III) oxidation state. The Values of Aa2, for which -6 x IO-3 A < Aa2 < 6 x Is -- 4p transition region is resolved into three transitions lo- A, are considered physically acceptable since Aa for having energy differences of -3 and -6 eV, respectively, liquids is -0.1 A. Values of AEo were restricted so that the indicating p,, py 0 pz. This is typical of distorted threshold energies for nitrogen and sulfur differed by no tetragonal (or distorted tetrahedral) coordination. In addi- more than 16.5 eV. This number is conservative and tion, a shoulder is observed between the Is - 3d and Is , somewhat arbitrary but is based on the largest difference 4p transitions that is likely a Is -, 4s transition. This latter in Eo found in our studies of copper model compounds (26). is classically "forbidden" but lack of inversion symmetry If this difference is larger than ±2 eV, the approximation can cause its appearance (32). that AEO is small in the fitting program is not valid, and this Upon reduction of the protein, the Is 3d transition is parameter becomes significantly correlated to other not observed, as expected for Cu(I). The Is -- 4p transi- parameters. When this occurs, the Eo used in the analysis tion region is resolved into two transitions having an energy of the model is appropriately changed by a few electron- difference of -7 eV, indicating pz < p, - py. This is volts, and the fitting program is rerun. Solutions could be indicative of trigonal or tetragonal geometry. However, the arbitrarily declared physically unacceptable based on very Is - 4s transition would not be as prominent for tetra- long or very short metal-ligand distances, but that situation gonal geometry as for trigonal symmetry, which suggests did not arise in these calculations. that the latter is most likely to occur for the reduced As described in Methods, loci of all physically accept- protein. able mathematical solutions were constructed and are Not until these edge features are completely understood shown in Fig. 6a. The boundaries of the loci are labeled as in terms of molecular orbitals or the site structure is to which criterion they violate. For example, in the upper modeled by well-characterized compounds can they be left and lower right regions of locus A, the difference between Eo for N and S becomes > 16.5 eV at the boundaries marked E. The other boundaries are caused by disorder parameters attaining computed nonphysical values. The exact boundary positions are somewhat dependent on convergence criteria of the nonlinear least-squares fitting program. However, the general shape and boundaries are representative. Surprisingly, one finds three over- lapping regions of fitted solutions, designated in Fig. 6 as A, B, and C, corresponding to convergence from widely differing initialization of the fitted variables. The rN and rs values, respectively, for each solution region of Fig. 6a are represented in the contours of loci B - -. . . ., -.- .. . . - . .. . . 4-0 690 9000 .:9020 and C where 1.90A - rN < 1.99A and 2.11 A rs '- X-RAY PHOTON ENERGY (eV) 2.22 A, with ±0.02 A error. Contours of TR? indicate FIGURE 5 X-ray absorption edge spectra of oxidized (-) and reduced that solution region C is not as good as those of A and B by (.* ) stellacyanin. A linear background has been subtracted to set the at least a factor of 2. Five solutions having integer values of absorption below the first transition equal to zero. NN and NS are contained in region A. The fit parameters

280 BIOPHYSICAL JOURNAL VOLUME 38 1982 .* -.N .: 1%s N.s. Ns FIGURE 6 Mathematical solutions to the EXAFS equation returned by the nonlinear fitting program for oxidized stellacyanin. (a) Three loci of physically acceptable solutions, A, B, and C. The boundaries are denoted by N± or S±, indicating that the disorder parameter for Nor S has become too large or too small (range ± 6 x 10-3 from models) and by E, indicating that the threshold energies ofNand S differed too greatly. J indicates an unstable jump to a more favorable solution. Note that the loci A, B, and C partially overlap the same region of parameter space. Contours show levels of equal MR,2. (b) Loci showing levels of equal Cu-N distance. (c) Loci showing levels of equal Cu-S distance. for these five solutions are given in Table I and will be that reduction might take place. For a 1 -mM sample of individually discussed below. protein, loss of color was observable at a rate as high as 0.25%/s at room temperature. Fig. 8 shows that the color Reduced State. For reduced stellacyanin, there loss that occurs at -500C after irradiation for the time is the same ambiguity in the assignment of chemical type required for four scans (a total of 28 min) is 7% of that of and number of ligands as for the oxidized protein. Fig. 7 the native state. However, the x-ray edge of this partially shows these results. Four solution regions (A, B, C, and D) bleached sample is not significantly shifted to lower ener- are distinguished, but only B and C contain both nitrogen gy, suggesting that color loss takes place without chemical and sulfur ligands. Regions A and D are actually line reduction of Cu(II), but arises from a loss of integrity of segments that describe only nitrogen or only sulfur liga- the blue copper site. At temperatures below -100°C we tion, respectively. Although ZR? is larger for A than for B find that color loss is greatly diminished so that samples or C, A contains only one-half the number of parameters, may be studied for 4 h without a detectable change of making it somewhat comparable to regions B and C in optical properties. Under these conditions trapped radicals "goodness of fit." Solution D may be discarded on the basis observed by EPR (24) accumulate to approximately the of its poor fit. Within region B three solutions having sample concentration (1 mM) with no obvious damage. integer values of NN and NS are found, and their fit Nevertheless, the two monitoring methods are used contin- parameters are summarized in Table I. uously and periodically, respectively, to ensure that sample changes of > -10% did not occur. A cautionary warning Radiation Damage. The effects of x-rays on arises from these studies. A sample irradiated at -100°C oxidized stellacyanin at temperatures between 250 and for 2 h at 1010 photons/s was warmed to the melting - 500C were monitored optically before, during, and after point, whereupon the accumulated trapped radicals drasti- the EXAFS experiment. As indicated previously (24), cally altered the protein, as indicated by optical bleaching x-rays produce hydrated electrons so that it was thought and presumably by other reactions as well. The effects of radiation at temperatures between 250 TABLE I and - 500C are also reflected in EXAFS data that were FIT PARAMETERS FOR THE PHYSICALLY ACCEPTABLE collected after the sample showed no further changes with SOLUTIONS TO THE EXAFS OF OXIDIZED (ox) AND time when in the x-ray beam. The EXAFS data, k3X(k), REDUCED (re) STELLACYANIN and Fourier transforms are shown in Figs. 9 and 10 for Solution rN rs Nao AEO 2:RI stellacyanin that was initially oxidized or reduced before radiation exposure at these temperatures. Fig. 11 com- oxINIS 1.92 2.20 4.3 x 10-3 5.0 x 10-3 0.1 1.41 pares the first-shell-filtered data with that collected at ox2NlS 1.96 2.21 -0.8 x 10-3 1.5 x 10-3 0.4 1.40 - ox3N lS 1.99 2.21 -3.1 x 10-3 -1.2 x 10-3 -0.2 1.78 temperatures of 1400C or below (cf. Fig. 4), where it was ox2N2S 1.96 2.18 -3.4 x 10-3 -1.8 x 10-3 -3.1 1.32 shown optically that there was little or no change on ox3N2S 1.99 2.17 -4.5 x 10-3 -3.3 x 10 -1.1 1.76 radiation exposure. The increase of amplitudes in Fig. 11 re2NOS 2.10 3.0 x 10-3 - 1.28 suggests that the radiation-damaged oxidized protein con- relNIS 2.07 2.31 5.6 x 10 -0.1 X 10-3 1.2 0.59 tains an extra ligand, probably sulfur. However, there are relN2S 2.07 2.26 5.1 x 10-3 -6.2 x 10-3 3.3 0.82 relN3S 2.06 2.22 4.9 x 10-3 -7.8 x 10-3 2.6 0.90 only small changes in the average distances to each of the ligand atoms. Analysis similar to that described above

PEISACH ET AL. Metal-binding Site ofStellacyanin Studied by EXAFS 281 EC

.EI

M(nm)

FIGURE 8 The apparent optical absorption spectra of a sample of stellacyanin (-3 mM as studied in the EXAFS sample holder):-, before x-ray exposure; and -, after four 7-min scans. Temperature during irradiation was - 500C and optical spectra were recorded at - 1300C with 2-mm optical path.

2.08 c x

... t i- --L -a 0 2.5 5 7.5 10 1.

FIGURE 9 Total EXAFS spectra (with background subtracted) multiplied by k' of the radiation-damaged stellacyanin in (a) the oxidized and (b) the reduced states. c

2.28 c

.A 2 34 NN 2.18 a. B .2 4 I w 2.20 2.22 a 4 2.28 -J uJ HD+2.22

ol Dio2.20- - - 0 I 2 4I b NS

ki FIGURE 7 Mathematical solutions to the EXAFS equation returned by 1.._ 0 2 4 6 8 10 the nonlinear fitting program for reduced stellacyanin. (a) Loci of r +a (K) physically acceptable solutions. The boundaries are denoted as in Fig. 6a. (b) Loci showing levels of equal Cu-N distance. (c) Loci showing levels FIGURE 10 Fourier-transformed data of radiation-damaged (a) oxi- of equal Cu-S distance. dized and (b) reduced stellacyanin.

282 ~~~~~~~~~~~~~~~BIOPHYSICAL JOURNAL VOLUME 38 1982 a

4- ..k b *. a .

FIGURE 12 X-ray absorption edge data of (a) oxidized ()and - -4 -. - damaged oxidized (. - ) and (b) reduced (-) and damaged reduced (. .v)stellacyanin. A linear background has been removed to set the K absorption below the first transition equal to zero. FIGURE II First-shell filtered data of (a) oxidized (-) and radiation- damaged oxidized (+) and (b) reduced (-) and radiation-damaged stellacyanin and the four acceptable solutions for reduced reduced (+) stellacyanin. stellacyanin will be discussed individually. The values of the parameters of the fit of each are listed in Table I. confirm that this is the case, with NN = 2, rN = 1.99 A, Solution oxI N IS has the physical disadvantage that, NS = 2, rs = 2.16 A. Similarly, it appears from Fig. 11 that with only two ligands, the x-ray edge and EPR spectra an additional ligand is present in the radiation-damaged would be difricult to explain. As noted above, the x-ray reduced protein, which in this case is probably a nitrogen edge suggests centrosymmetry; the EPR spectrum is rhom- atom, and that the average ligand distance is now larger. In bic and is unlikely to arise from bidentate coordination. fact, analysis shows NN = 2, rN = 2.08 A with NS = 2, rs = Also, two-atom coordination implies a most unusual chem- 2.31 A. The distance error in both redox states is ±0.03 A. istry for Cu(II), which in most instances is found with four The ligand distances in neither redox state of damaged near ligands. stellacyanin are similar to those found in undamaged Solution ox2N 15 has the disorder parameters that one reduced stellacyanin or in either of the reduced states would expect, relative to the model compounds. The reported for plastocyanin (15, 34) as might have been Cu-N distance is slightly longer than in the model and expected if reduction by hydrated electrons were the only slightly more disordered; the Cu--S distance is shorter process to have occurred. Previously, Chance et al. (24) than the model and more ordered. The three near ligands reported similar observations with cytochrome c oxidase; would be somewhat analogous to the copper site in oxidized reduction by hydrated electrons takes place, accompanied plastocyanin and azurin and could give rise to the observed by structural changes unlike those caused by chemical x-ray edge and EPR spectra. reduction. Solution ox3N IS has a more disordered Cu-S bond Fig. 12 compares x-ray edge absorption data for radia- than the model, although it's length is shorter. Th-e fit is not tion-damaged protein with those for the undamaged pro- as good as that for ox2N IlS. While, in principle, one sulfur tein (Fig. 5). The data for damaged and undamaged atom could be formally 5- with the other 5o, this would oxidized proteins are nearly identical with the only differ- lead to a very large disparity in Cu-S distances, which ences being a shift of -I eV of the Is - 3d transition to would appear as a greatly disordered metal-ligand bond. lower energy and a loss of resolution in the Is - 4s region. Solution ox3N2S provides for more disorder in both the For the oxidized damaged protein, the structural change Cu-N and Cu-S bonds than in the models. Five close that takes place (probably the addition of a second sulfur neighbors to Cu(II) is an unusual situation except in atom to the first coordination shell at the same distance as porphyrins and in square pyramidal complexes supported the original one) apparently alters the geometry of the by macrocycles. The fit is not as good as that for ox2N 15. copper site only slightly. Solution re2N0S provides the worst fit of the various solutions for the reduced but as there are DISCUSSION plausible protein, few variable fitting parameters, this is not so disadvanta- The five mathematically and physicaHly acceptable solu- geous as it may first seem, and we cannot discount this tions with integral values of NN and Ns for oxidized solution entirely-

PEISACH ET AL. Metal-binding Site ofSzellayanin Studied by EXAFS 283 Solution reiN IS shows a much more ordered Cu-N giving an average distance of 2.82 ±+ 0.03 A, but, the bond in the protein than in the model in spite of the fact fourth shell of CuTPP was also a likely candidate, giving that its distance is longer, but this puzzle is present in all an average distance of 2.95 ±+ 0.03 A. The possibility that three sulfur-containing solutions for the reduced protein. this shell is a sum of contributions from different atoms is Solutions relN2S and relN3S show disordered Cu-S likely, but without information as to the chemical types of bonds and both require quite large shifts in AEo of the contributing atoms, further analysis is futile. model compounds in order to fit the protein EXAFS data. Both Figs. 6 and 7 contain a point labeled HD. This When the models were reanalyzed by shifting their own designates the solutions for both the oxidized and reduced AEo in a compensating direction before preparing the proteins that are arrived at by the Hodgson-Doniach derived model amplitudes and phases, the resulting solu- method (35). This method uses the total EXAFS data tions for the protein required smaller shifts (as expected) (similar to that shown in Fig. 2) which contains the sum of but resulted in even more disorder in the Cu-S bonds, contributions of all shells, noise, and possibly residual even exceeding our physically acceptable limits. This indi- contributions due to insufficient background removal. cates that part of the goodness of fit of these two solutions These data are then fit by comparison with models, and no may be attributed to correlations between Eo and U2 rather change is allowed for the At2 or AEo contributions (i.e., than to a good correspondence between the theoretical A.X2 = AEo = 0). The problems and possible pitfalls of this model and the molecule. If either of these solutions is approach are demonstrated in Figs. 6 and-7 together with chosen to represent that for the reduced protein, it would reported results for azurin (36) and other proteins (37). be suggestive that the sulfur atoms are quite inequivalent, Detailed discussions of analytical methods are given by even though they are all in the first coordination sphere. Lee et al. (27) and Powers (28). In summary, the ox2NiS solution seems the most That portion of this work carried out at the Albert Einstein College of favorable for the oxidized protein but not by an over- Medicine was supported by a U.S. Public Health Service grant HL-13399 whelmingly convincing margin. For the reduced state, one to Dr. Peisach and as such is Communication 423 from the Joan and can say that the metal site most likely contains only one Lester Avnet Institute of Molecular Biology. Part ofthis work was done at nitrogen atom and very possibly only one sulfur atom. If the Stanford Synchrotron Radiation Laboratory under Proposal No. there is more than one sulfur atom present, then they must 105/423 and 424. The portion of this work carried out at the University of Pennsylvania was supported in part by U.S. Public Health Service grants be quite inequivalent to provide the observed disorder. GM-27308, GM-28385, HL-15061, and HL-18708 to Dr. B. Chance. Relationship to Other Structural Parame- Receivedfor publication 2 October 1981 and in revisedform 8 January ters. Recently, the crystal structure of reduced plasto- 1982. cyanin has been reported and a high and a low pH form found (34). The high pH or redox active form contains one REFERENCES sulfur (2.2 A) and two nitrogen atoms (2.1, 2.3 A) in the shell of the atom with the 1. Malkin, R., B. G. Malmstrom. 1970. The state and function of first coordination copper copper in biological systems. Adv. 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