EPR of azurins from pseudomonas aeruginosa and alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in pseudomonas aeruginosa protein

EPR of Azurins from Pseudomonas aeruginosa and Alcaligenes denitrificans Demonstrates pH-Dependence of the Copper-site Geometry in Pseudomonas aerzginosa Protein C. M. Groeneveld, R. Aasa, B. Reinhammar, and G. W. Canters CG, GC. Gorlaeus Laboratories, Leiden University, The Netherlands..-RA, BR. Department of Biochemistry and Biophysics, Chalmers University of Technology, Giiteborg, Sweden.

ABSTRACT The X- and Q-band

EPR spectra of Pseudomonas aeruginosa (63Cu)azurin and Alcaligenes azurin have been measured at pH = 5.2 and 9.2, in the presence and absence of 40% glycerol. The EPR spectra of both proteins could properly be simulated by taking into account a spread in the tetrahedral angle of the copper site. The change in the EPR spectrum of Pseudomonas aeruginosa (63Cu)azurin that is observed upon an increase of the pH from 5.2 to 9.2 is consistent with a small decrease of the average tetrahedral angle from 61 o to 60”. This geometrical change is consistent with the interpretation of earlier NMR and EXAFS observations. No pH effect is observed for Alcaligenes denitrifcans azurin, in agreement with predictions based on crystallographic evidence. Glycerol has only a marginal effect on the appearance of the EPR spectra, and does not alleviate the “g-strain.”

denitrifcans

INTRODUCTION The pH dependence of the conformation of a number of blue-copper proteins is only partly understood, and still intrigues many researchers. The redox center in these relatively small (AZ = lo-20 kDa) electron-transfer proteins consists of a single copper ion coordinated by two nitrogens from histidine residues and two sulfurs, which usually derive from a methionine and a cysteine [l-8]. In the plastocyanins-the Address reprint requests to G. W. Canters, Gorlaeus Laboratories, 2300 RA Leiden, THE NETHERLANDS.

Leiden University,

Journal of Inorganic Biochemistry 31, 143-154 (1987) 0 1987 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017

P. 0. BOX 9502, 143 0162-0134/87/$3.50

144 C. Groeneveld et al.

blue-copper

proteins responsible for the shuttling of electrons between the cytochrome and photosystem I in chloroplasts-one of these histidines becomes protonated at low pH ( pKO - 6), whereby it becomes detached from the copper and leaves the metal in a three-coordinated environment [8, 9].The latter coordination strongly favors the Cu(I) form over the Cu(II) form, and essentially leads to a switching off of the redox activity of the plastocyanin [8, 91. No such pH effects are observed for the (bacterial) azurins as a class in general. However, in the particular case of azurin from Pseudomonas aeruginosa, the redox activity of the protein towards cytochrome-cs5r has been reported to increase by at least two orders of magnitude when the pH is lowered from 9 to 5 [lo], although, recently, some of the evidence has been disputed [ 11, 121. The origin of this pH effect is different from that observed for the plastocyanins, as spectroscopic studies have established that it originates from the protonation/deprotonation of a histidine residue (His-35). This residue is not a copper ligand but is situated in the second coordination sphere of the copper next to ligand His-46 [13-161. A change in the state of protonation of His-35 leads to a reorientation of this residue and a concomitant change of protein conformation, in particular in the neighborhood of the copper-site [13]. For some time it was thought that this change in the first and/or second coordination shell of the copper would bear directly on the protein’s redox activity [ 161. Recently it has been found, however, that the electron self-exchange rate of P. aeruginosa azurin is independent of pH in the range of 5 c pH < 9 [ 17, 181. This observation, together with the purported pH dependence of the azurin/cytochrome-cssl electron exchange kinetics, has been considered evidence that different parts of the protein are involved in the two electron transfer reactions [ 17-221. Still, the question remains as to what exactly happens with the copper-site geometry when the pH is changed. Proton NMR experiments indicate changes of the order of 0.15-l A [13] in the positions of the ligand methionine, His-35, and at least one ligand histidine, while, on the other hand, EXAFS measurements show the copp!ligand distances to be unaffected by pH within the accuracy of the experiment (0.03 A) [23]. To further map the pH dependence of the copper-site geometry, low temperature (40 K) EPR experiments have now been performed on solutions of azurin from P. aemginosa at high (9.2) and low (5.2) pH. To obtain optimum resolution, isotopically pure (63Cu)azurin was employed. On the basis of crystallographic studies, the structure of the closely related azurin from Alcaligens denitrificans has been predicted to be insensitive to pH [3]. This is because the cleft in which His-35 resides is accessible to solvent in P. aeruginosa azurin, but is virtually closed off from the outside medium in A. denitrzfcans azurin [3]. Preliminary l H-NMR results of this protein appear to confirm this expectation (unpublished results). For comparison, therefore, the EPR spectrum of the latter azurin was also recorded, at high (9.2) and low (5.2) pH. Since resolution was considered less critical in this case, native protein (69 % 63Cu and 3 1% 65Cu) was used. Finally, there are reports in the literature that a possible tendency of P. aeruginosa azurin to form aggregates towards low temperatures [24] may be counteracted by glycerol. To check this possibility, experiments were also performed on protein solutions to which glycerol (40%) was added. The results are reported here. One of the findings is that a distribution of magnetic parameters (811- and Ali values) is required to explain the appearance of the 811region of the EPR spectra (“g-strain”). Furthermore, the high- and low-pH forms of P. aeruginosa azurin differ slightly, but significantly; the difference can be related to a small change in the average ligand-copper-ligand angles.

f/b6 complex

PSEUDOMONAS

MATERIAL

AERUNGINOSA

PROTEIN pH-DEPENDENCE

145

AND METHODS

Protein Preparation The growth of P. aeruginosa cultures and the isolation of the azurin were performed as described by Ambler [25] and Parr et al. [26]. The preparation of the apoprotein and its reconstitution with @Cu (commercially available as metal from Inter-sales Holland B.V.) was performed as described earlier [27]. A. denitrificans bacterial paste was kindly provided by Dr. R. P. Ambler, and the azurin was isolated according to the procedure described by Norris et al. [4]. The purity of both azurins was checked by measuring the ratio of the optical absorbances at 625 nm and 280 nm, which amounted to 0.56 and 0.30 for, respectively, P. aeruginosa and A. denitrificans azurin. The azurin solutions were made up in 20 mM MES buffer (MES = 2-[Nmorpholino]ethanesulfonic acid), pH = 5.2 oi in 20 mM borate buffer, pH = 9.2, by ultrafiltration in Amicon equipment. EPR Measurements The X-band EPR spectra were recorded on a Bruker ER 2OOD-SRC EPR spectrometer, which was interfaced to an ASPECT-2000 computer and equipped with an Oxford Instruments ESR-9 helium-flow cryostat. The X-band spectra were measured at magnetic fields between 0.275 T and 0.355 T, with a modulation amplitude of 0.5-1.0 mT, a microwave power of 0.2 mW, and at a frequency of 9.46 GHz. The measurements at Q-band were made with a Varian model V-4503 spectrometer console and a homebuilt helium cryostat. The output from the V-4560 100 kHz modulation unit was fed via a differential amplifier to a time base ER 001 of the Bruker console, and to the computer interface ER 144 C of the Aspect computer. The Q-band spectra were measured at fields of between 1.04 T and 1.24 T, with a modulation amplitude of 2.0 mT, a microwave power of about 0.1 mW, and at a frequency of around 34.4 GHz. Both X- and Q-band EPR spectra were recorded at about 30 K. X-band klystron frequencies were measured with an HP model 5245 L electronic counter, while Q-band frequencies were determined from the known g value, 2.0036, of cr,a!‘diphenyl-P-picrylhydrazyl (DPPH). EPR Analysis EPR spectra were simulated with the program ANGAWA, using a second-order perturbation calculation, which was originally developed by Ammeter and coworkers (Zurich) [ZS], and was adapted by P.E.M. Wijnands and M. H. Welter at the Chemistry Department in Leiden. Further details are given in the Results section. RESULTS The X- and Q-band EPR spectra of P. aeruginosa (63Cu)azurin and native A. denitrificans azurin were measured at pH = 5.2 and 9.2, in the presence (40% v/v) and absence of glycerol. The experimental EPR spectra of P. aeruginosa (63Cu)azurin (pH = 5.2, no glycerol) and their simulations are shown in Figure 1 (X-band) and Figure 2 (Q-band). Those of A. denitrifcans azurin @H = 9.2, no glycerol) are shown in Figure 3 (X-band) and Figure 4 (Q-band). As is obvious from the gI region in the X- and Q-band EPR spectra of both proteins, straightforward simulations of the EPR spectra using only gll, g, , All, Al, and linewidth as adjustable parameters (denoted by c’ (X&band) and c (Q-band) in the

146 C. Groeneveld et al.

I-

1

0.35

0.30 magnetic

field

IT)

FIGURE 1. X-band EPR spectrum of 1.2 mM P. aeruginosa (63Cu)azwinin 20 m&i MES buffer at pH = 5.2, without glycerol, measured at 30 K. Other EPR conditions: see Material and Methods section; (a) experimental EPR spectrum; (b) computer simulation with A@’ = 1.2”) a linewidth of 30 G and g and A values as reported in Table 1. Inset: enlargement of the gn region; (a’) experimental EPR spectrum; (b’ ) computer simulation as (a); (c ’ ) computer simulation as (b) but with Afi ’ = 0”.

figures) da not agree very well with experiment. While the X-band simulations show four equally

spread,

equally

well-resolved

hyperfine

lines

in the 811 region,

the

experimentally ObSeNed hyperfine lines exhibit different widths and spacings. Observations of this kind are not unconunon for proteins with pammagnetic centers like blue-copper proteins [29, 301, heme proteins [31], and iron-sulfur proteins [32]; they have been captured under the heading of so-called g-strain effects. In practice, this amounts to the assumptibn that the EPR spectrum is not characterized by a single set of EPR parameters, but by a distribution of such sets. Brill[29] has been the first to analyze this effect successfully for the blue-copper proteins, and his approach is adopted here. Starting point for the analysis is the use of an (idealized) Dzd symmetry for the description of the copper center. Brill showed that successful simulations of the experimental EPR spectra are possible when the variation in EPR parameters referred to above is related to a distribution in the value of a single geometrical parameter,

PSEUDOMONAS AERUNGZNOSA PROTEIN pH-DEPENDENCE

I

1.05

-

’

-

’

1

”

8

’

1.10

1

“S

c

-

-

1.20

1.15 magnetic

FIGURE

1

147

field(T)

2. Q-band EPR spectrum of 1.2 mM P. ueruginosu(63Cu)azurinin 20 mM MES

buffer at pH = 5.2, without glycerol, measured at 30 K. Other EPR conditions: see Material and Methods section; (a) experimental EPR spectrum; (b) computer simulation with A@’ = 1.2”) a linewidth of 50 G and g and A values as reported in Table 1; (c) computer simulation as (b), but with A/3’ = 0“.

namely the tetrahedral angle 0, depicted in Fig. 5. It is well-known that small variations in /3 mainly affect the g11- and AlIvalues [29,33-361; when adopting Brill’s approach, g, and A, will, therefore, be kept constant (for A,, a value of O.lAll is used 129, 37J), and only the dependence of gl and A,, on 0 has to be established. Solomon and coworkers [33, 341 have shown that a ligand-field theoretical treatment of the copper center according to Dz,~symmetry leads to the following dependence of 811on P: g,,=2--

sin4/3

in which c contains a number of atomic and molecular parameters. Instead of employing the theoretical expression of c, a semi-empirical value is obtained as follows. According to Solomon, the best fit between the optical characteristics of the blue-copper centers and the ligand-field model is obtained for values of p around 60” [33]. Taking as a reference point the highest gll-value found in this study (811= 2.276 for P. aeruginosa (63Cu)azurin at pH = 9.2, no glycerol), and equating this to expression (1) with 0 = 60”, one finds c = - 0.16. Although this way of tying up P with the gl scale is somewhat arbitrary, the present analysis of the strain effects hinges on the variation of gll and Ali with 0, and within the range of presently relevant P-

148

C. Groeneveld

et al.

a

b

I

I

I

I

I

I

0.30

1

035 magnetic

field

(T)

FIGURE 3. X-band EPR spectrum of 0.7 mh4 A. denitrifcans native azurin in 20 mh4 borate buffer at pH = 9.2, without glycerol, measured at 30 K; (a) experimental EPR spectrum. (b) computer simulation with A/S’ = 1.O”, a linewidth of 34 G and g and A values as reported in Table 1. Inset: enlargement of the gll region; (a’) experimental EPR spectrum; (b’) computer simulation as (b); (c’) computer simulation as (b), but with A/3’ = 0”.

values, this variation is not very sensitive to the choice of the reference point on the gll-versus p scale. To establish the dependence of All (the sign of Ali is disregarded in the present treatment) on P, use is made of the empirical observation that for copper-coordination compounds [35, 381 and blue-copper centers [39], the values of the A- and g-tensor usually exhibit a good linear correlation leading to an expression of the form

A,,=A,,“+cr

dgll(P-PO)

dp

The value of the proportionality constant (Ywas determined empirically in the present case on the basis of the first-stage simulations of the EPR spectra and amounted to cy = 0.046 cm-t. Again, the outcome of the present analysis is not very sensitive to the

PSEUDOMONAS

1.05

l.iO

AERUNGINOSA

li5

PROTEIN pH-DEPENDENCE

149

l.iO mognetlc

held ITI

FIGURE 4. Q-band EPR spectrum of 0.7 mM A. denitrificans native azurin in 20 mM borate buffer at pH = 9.2, without glycerol, measured at 30 K. Other EPR conditions: see Material and Methods section; (a) experimental EPR spectrum; @) computer simulation with A/3’ = l.O”, a linewidth of 50 G and g and A values as reported in Table 1; (c) computer simulation as (b), but with A@’ = 0”.

precise value of this constant. The spread in &values was accounted Gaussian distribution of fl around an average value Bo, according to

WC& A@‘>=

’

A/34%

&wo)2~2(4‘J~)2

for by using a

(3)

EPR spectra were obtained as summations of individual EPR spectra calculated for a set of discrete B-values, each spectrum being given its proper weight according to equation (3). The summations appeared to converge rapidly, and, in practice, summation of between five and nine spectra proved sufficient. Values of Allo, /30 and A/3 ’ were varied until visual agreement between theory and experiment was obtained. The final procedure has been as follows. The high-field region of the Q-band EPR spectrum was simulated to obtain values of g, and gu. Subsequently, the X-band EPR spectrum was simulated, and optimum values of Aljo, POand A/3 ’ were determined. 2 b

FIGURE 5. Definition of the tetrahedral angle /3 in a reference coordinate system used for the flattened tetrahedral (Dw) ligand-field model. Ligands are denoted by L.

L

‘Y

150

C. Groeneveld

TABLE

et al.

1. Parameters Used to Simulate the X- and Q-band EPR Spectra of P. aeruginosu (63Cu)Azurin and Native A. denitrificans Azurin as Measured Under Various Conditions”

gub

gzc

Allo x 104, cm-’

A@’

2.035

2.052

2.263

56

1.2

61.0

0 0

2.035 2.035 2.034

2.051 2.051 2.050

2.260 2.276 2.258

58 51 62

1.0 1.0 0.8

61.3 60.0 61.5

0

2.032

2.049

2.254

62

1 .o

61.9

Glycerol %

Species

PH

Pseudomonas

5.2

0 40

9.2 5.2 9.2

gx

b

PO

aeruginosad

Alcaligenes denitrifican.P

0 in all simulations A I was set equal to 0.1 All. b obtained from the Q-band EPR spectra; estimated accuracy: ’ obtained from the X-band EPR spectra; estimated accuracy: d 63Cu)azurin. p native azurin.

Finally, with spectrum was b’ (X-band) obtained from

k 0.002. * 0.002.

the values of 811, g,, g,, ~$1o and Ap ’ , the complete Q-band EPR simulated as a check. The final results of the simulations are denoted by and b (Q-band) in Figures l-4; the values of the EPR parameters the analysis are presented in Table 1.

DISCUSSION The Q-band EPR spectra of both the P. aeruginosa and the A. denitrificans azurin demonstrate the presence of a rhombic component of the g-tensor. Similar observations have been reported by Brill for azurin [29], and by Solomon and coworkers for plastocyanin [40]. In these studies, as in the present one, the anisotropy amounts to about Ag = 0.017. On theoretical grounds, in the case of plastocyanin, this relatively large rhombicity has been considered indicative of strong delocalization (35 %) of the unpaired electron over the 3p orbital of the ligand cysteine-sulfur. If this inference is correct, the present observations may indicate that such strong delocalization is a more general feature of the type-1 copper site in azurins [24], plastocyanins [40], and laccases [4 11. It is noteworthy that for both azurins studied here, the X-band as well as the Q-band EPR spectrum can be simulated by the same set of magnetic parameters (including AD ’ , but with an exception for the line-width). Even the slightiy skewed appearance of the gll region in the Q-band EPR spectrum of the P. aeruginosa azurin is faithfully reproduced by the simulation (Fig. 2; lower S/N precludes a similar conclusion for the Q-band EPR spectrum of the A. denitrijicans azurin). Surprisingly, the distribution of magnetic parameters can be characterized by a single structural parameter, namely, the spread in the tetrahedral angle 0. The correlation between 811and Ali gives the gll region in the X-band EPR spectrum its characteristic appearance. The correlated changes in 811and Ali approximately cancel at the low-field hyperfine component, but add up for the high-field one, leading to a smearing out of the latter. In view of the stronger structural constraints imposed on His-35 in the A.

PSEUDOMONAS

AER UNGINOSA PROTEIN pH-DEPENDENCE

151

denitrificans azurin, the conformational variability of the copper site in this protein might be expected to be somewhat smaller than for P. aeruginosa azurin. The accuracy of Afi' , reported in Table 1, is insufficient, however, to decide whether the difference of 20% between the Afl’-values of the two proteins is significant. The data of the present EPR study on A. denitrificans azurin agree roughly with those reported by Baker et al. [42], although the latter authors did not take “g-strain” into account nor did they perform Q-band experiments. The latter point may explain why their g, value deviates from the average of the g,- and g,,-values found here. For A. denitrificans azurin, no pH effect is observed on the EPR spectrum. We have checked whether a possible effect might have been obscured by the use of copper of natural isotopic composition. It appears, however, that the EPR spectrum of such a species can be satisfactorily simulated by adjusting the line-width slightly (5%). The conclusion must, therefore, be that there is no pH effect on the copper-site geometry in A. denitrificans azurin, in accordance with predictions from crystallographic studies [3] (The pH effect observed by others [42] occurs only at pH > 11). In contrast to the A. denitrificans case, the EPR spectrum of azurin from P. aeruginosa exhibits a clearly distinguishable pH effect, as is shown in Figure 6. The gl and Ali values change from 2.263 and 56 x 10m4cm-i to 2.276 and 51 x 10m4 cm-’ when the pH is increased from 5.2 to 9.2, corresponding with a one degree decrease in &. On the other hand, the spread in the distribution of /3 appears not affected by pH, the rms-value of the distribution staying around lo. With a bond length of about 2 A [l, 2, 231, the change in PO would correspond to a lateral displacement of the ligand atoms of 0.035 A. Addition of glycerol has no effect on the EPR spectra of the A. denitrificans azurin (not shown). In contrast, the P. aeruginosa azurin EPR spectrum at high pH changes significantly when glycerol is added (see Fig. 6), and takes on the features of the lowpH form. This was found to be a trivial effect, however, since it is due to the sensitivity of borate buffer to polyols [43]; addition of 40% glycerol in the present case appears to lower the pH by about 4 pH-units. The glycerol effect on the low-pH EPR spectra of P. aeruginosa azurin (measured in the glycerol insensitive MES buffer) is only marginal (see Figs. 6A and 6B), and can be accounted for by a slightly larger /30 value and concomitant changes in 811and Allo (see Table 1). Although the pH of MES buffer has a small temperature coefficient, the glycerol effect might nevertheless be real and due to a slight change in effective pH during freezing of the solution. Contrary to what has been suggested elsewhere [31], the presence of glycerol does not seem to reduce the “g-strain,” since Afl’ appears unaffected by glycerol. The present experiments do not allow for a conclusion concerning the static or dynamic character of the spread in 0. Certainly, the amplitude of the ligand displacement found here is within the range calculated for atomic displacements in proteins [&l-46]. CLOSING REMARKS To what extent can the present findings for the P. aeruginosa azurin be reconciled with the results of earlier NMR and EXAFS experiments? Is it possible now to obtain a coherent picture of the fluxionality of the copper site? From ’ H-NMR experiments of the reduced protein it has transpired that the high- and low-pH forms of the azurin exhibit at least the following differences [ 13, 16,471: (a) a difference of about 1 A in the distance of the His-35 &-proton to the copper atom; (b) a difference of at least 0.15 A in the average position of the +CH3 group of the ligand Met-121 with respect

152

C. Groeneveld

et al.

-

mognetlc

field

IT)

mognetlc

magnetac

field

(Tl

magnettc

his

field

ITI

o.io fmld

IT1

Enlargement of the 811region of the X-band EPR spectra of P. aemginosa (63Cu)azurin as a function of pH and amount of glycerol; traces marked (a) denote experimental EPR spectra; traces marked (b) the computed EPR spectra. Panel A: pH = 5.2, no glycerol; A/3’ = 1.2”. Panel B: pH = 5.2,40% glycerol, A/3’ = 1.0”. Panel C: pH = 9.2, no glycerol, AD’ = 1.0”. Panel D: pH = 9.2, 40% glycerol, A@’ = 1.0”. Parameters used in tbe simulations are given in Table 1. EPR spectra at pH = 5.2 were obtained in 20 mM MES buffer, EPR spectra at pH = 9.2 in 20 mM borate buffer. Other experimental conditions as described in the captions of Figures 1 and 2, and as given in the Material and Methods section. FIGURE

6.

ring of Phe-15; (c) a difference of 0.5-0.9 A of the His-46 C4-proton with respect to the His-35 ring. It cannot be concluded from the NMR experiments, however, whether these changes in distance reflect alterations in the copper coordination, a reorganization of the secondary-coordination shell of the metal only, or both. On the other hand, EXAFS experiments indicate that the copper-ligand distances are almost unaffected by pH (within 0.03 A). Finally, the results of the present EPR study are compatible with pH-induced lateral displacements of the ligands with respect of the copper center of the order of 0.03 A. The structural changes in the first-coordination shell are, evidently, much smaller than the conformational changes estimated on the basis of the NMR experiments. It is clear that the copper-coordination geometry remains relatively unaffected by pH, and the effects observed by NMR must, therefore, originate from structural changes in the second-coordination sphere of the copper, possibly combined with a change in the position and orientation of the

to the aromatic

PSEUDOMONAS AERUNGINOSA

PROTEIN pH-DEPENDENCE

153

metal plus its first-coordination shell with respect to the protein surrounding. It has been suggested that the peculiar structure of the copper-site in type-1 blue-copper proteins is dictated by the protein environment, and not so much by the metal atom [7, 481. If this statement is correct, it is an intriguing question how the protein manages to maintain the integrity of the copper site while reshuffling its own structure. The authors thank Dr. R. P. Ambler for providing them with bacterial paste of A. denitrificans, Mr. P. E. M. W@ands for his help with the EPR simulations, and Professor Dr. J. Reedijk for critical reading of the manuscript. This work was supported by grants from the Netherlands Organization for the Advancement of Pure Research (Z. W.O.) and from the Swedish Natural Research Council.

REFERENCES 1. E. T. Adman, R. E. Stenkamp, L. C. Sieker, and L. H. Jensen, J. Mol. Biol. 123, 3547 (1979). 2. E. T. Adman and L. H. Jensen, Zsr. J. Chem. 21, 8-12 (1981). 3. E. T. Adman, in Topics in molecular and structural biology. Metalloproteins., P. Harrison, Ed., VCH Verlagsgesellschaft, Weinheim, BRD, 1984, Vol. 1, pp. 1-41. 4. G. E. Norris, B. F. Anderson, E. N. Baker, and S. V. Rumball, J. Mol. Biol. 135, 309-312 (1979). 5. G. E. Norris, B. F. Anderson, and E. N. Baker, J. Mol. Biol. 165, 501-521 (1983); J. Am. Chem. Sot. 108, 2784-2785 (1986). 6. P. M. Colman, H. C. Freeman, J. M. Guss, M. Murata, V. A. Norris, J. A. M. Ramshaw, and M. P. Venkatappa, Nature 272, 319-324 (1978). 7. T. P. J. Garrett, D. J. Clingeleffer, J. M. Guss, S. J. Rogers, and H. C. Freeman, J. Biol. Chem. 259, 2822-2825 (1984). 8. J. M. Guss, P. R. Harrowell, M. Murata, V. A. Norris, and H. C. Freeman, J. Mol. Biol. 192, 361-387 (1986). 9. H. C. Freeman, in Coordination Chemistry, J. P. Laurent, Ed., Pergamon Press, Oxford, 1981, Vol. 21, pp. 29-51. 10. M. C. Silvestrini, M. Brunori, T. Wilson, and V. Darley-Usmar, J. Znorg. Biochem. 14, 327-338 (1981). 11. A. F. Corm, R. Bersohn, and P. E. Cole, Biochemistry 22, 2032-2038 (1983). 12. 0. Farver and I. Pecht, in Copper Proteins and Copper Enzymes, R. Lontie, Ed., CRC Press, Florida, 1984, Vol. 1, pp. 183-215. 13. E. T. Adman, G. W. Canters, H. A. 0. Hill, and N. AKitchen, FEBS Lett. 143, 287-292 (1982); Znorg. Chim. Acta 79, 127-128 (1983). 14. K. Ugurbil and R. Bersohn, Biochemistry 16, 3016-3023 (1977). 15. H. A. 0. Hill and B. E. Smith, J. Znorg. Biochem. 11, 79-93 (1979). 16. G. k. Canters, H. A. 0. Hill, N. A. Kitchen, and E. T. Adman, Eur. J. Biochem. 138, 141-152 (1984). 17. C. M. Groeneveld and G. W. Canters, Rev. Port. Quim. 27, 145 (1985). 18. C. M. Groeneveld and G. W. Canters, Eur. J. Biochem. 153, 559-564 (1985). 19. A. G. Lappin, M. G. Segall, D. C. Weatherbum, R. A. Henderson, and A. G. Skyes, J. Am. Chem. Sot. 101, 2302-2306 (1979). 20. 0. Farver and I. Pecht, Zsr. J. Chem. 21, 13-17 (1981). 21. 0. Farver, Y. Blat& and I. Pecht, Biochemistry 21, 3556-3561 (1982). 22. I. Pecht, 0. Farver, and A. Licht, Rev. Port. Quim. 27, 45-46 (1985). 23. C. M. Groeneveld, M. C. Feiters, S. S. Hasnain, J. van Rijn, J. Reedijk, and G. W. Canters, Biochim. Biophys. Acta 873, 214-227 (1986).

154

C. Groeneveld

et al.

24. W. B. Mims and J. Peisach, in Biological Magnetic Resonance, L. J. Berliner and J. Reuben, Eds., Plenum Press, New York, London, 1981, Vol. 3, Chap. 2. 25. R. P. Ambler, Biochem. J. 89, 341-349 (1963). 26. S. R. Parr, D. Barber, C. Greenwood, B. W. Phillips, and J. Melling, Biochem.

J.

157, 423-430 (1976). 27. C. M. Groeneveld, S. Dahlin, B. Reinhammar,

and G. W. Canters, J. Am. Chem. Sec. 109, 3247-3250 (1987). 28. C. Daul, C. W. Schlopfer, B. Mohos, J. Ammeter, and E. Gamp, Comp. Phys. Commun. 21, 385-393 (1981). 29. A. S. Brill, in Transition Metals in Biochemistry, Springer-Verlag, New York, 1977, pp. 40-80. 30. C. X. Wang, G. Giugliarelli, S. Cannistraro, and C. Fini, NUOVO Cimento D 8, 76-90

(1986). 31. W. R. Hagen, J. Magn. Res. 44, 447-469 (1981). 32. D. 0. Hearshen, W. R. Hagen, R. H. Sands, H. J. Grande, H. L. Crespi, I. C. Gunsalus, and W. R. Dunham, J. Magn. Res. 69, 440-459 (1986). 33. E. I. Solomon, J. W. Hare, D. M. Dooley, J. H. Dawson, P. J. Stephens, and H. B. Gray, J. Am. Chem.Soc. 102, 168-178 (1980). 34. H. B. Gray and E. I. Solomon, in Copper proteins; Metal Ions in Biology. T. G. Spiro, Ed., Wiley, New York, 1981, pp. 2-39. 35. A. W. Addison in Copper Coordination Chemistry; Biochemical and Inorganic Perspectives, K. D. Karlin and J. Zubieta, Eds., Adenine Press, New York, 1983, pp. 109-128. 36. T. Vtingird in Biological Application of Electron Spin Resonance, H. M. Swarm, J. R. Bolton, and D. C. Borg, Eds., Wiley-Interscience, New York, 1972, pp. 411-447. 37. A. S. Brill and G. F. Bryce, J. Chem. Phys. 48, 43984404 (1968). 38. H. Yokoi and A. W. Addison, Inorg. Chem. 16, 1341-1349 (1977). 39. J. Peisach and W. E. Blumberg, Arch. Biochem. Biophys. 165, 691-708 (1974). 40. K. W. Penfield, A. A. Gewirth, and E. I. Solomon, J. Am. Chem. Sot. 107,45194529 (1985). 41. B. Reinbammar, in The Coordination Chemistry of Metalloenzymes, I. Bertini, R. S. Drago, and C. Luchinat, Eds., D. Reidel Publishing Company, 1983, pp. 177-200. 42. E. W. Ainscough, A. G. Bingham, A. M. Brodie, W. R. Ellis, H. B. Grat, T. M. Loehr, J. E. Plowman, G. E. Norris, and E. N. Baker, Biochemistry 26, 71-83

(1987). 43. D. D. Perrin and B. Dempsey, 44.

45. 46. 47. 48.

in Buffers for pH and Metal Ion Control, Fletcher and Son Ltd., Norwich, Great Britain, 1974, pp. 55-61. H. Frauenfelder, G. A. Petsko, and D. Tsernoglou, Nature 280, 558-563 (1979). H. Hartmann, F. Parak, W. Steigemann, G. A. Petsko, D. R. Ponzi, and H. Frauenfelder, Proc. Natl. Acad. Sci. USA 79, 4967-4971 (1982). B. Brooks and M. Karplus, Proc. Natl. Acad. Sci. 80, 6571-6575 (1983). C. M. Groeneveld and G. W. Canters, J. Biol. Chemistry, submitted (1987). H. G. Gray and B. G. Malmstriim, Comments Inorg. Chem. 2, 203-209 (1983).

Received May 8, 1987; accepted May 28, 1987