The proton spin-flip lines of Mo(V) EPR signals from sulfite oxidase and xanthine oxidase

JOURNAL

OF MAGNETIC

RESONANCE

64,384-394

(1985)

The Proton Spin-Flip Lines of Ma(V) EPR Signalsfrom Sulfite Oxidase and Xanthine Oxidase GRAHAM School

of Chemistry

NEIL GEORGE

and Molecular Sciences, University Brighton BNl 9QJ, United Kingdom

of Sussex,

Falmer,

Received December 26, 1984; revised May 2, 1985 The proton spin-flip transitions in MO(V) EPR spectra of the different reduced forms of the enzymes xanthine oxidase and sulfite oxidase have been examined. The proton spin-hip transitions of xanthine oxidase originate from weakly coupled nonexchangeable nuclei, probably carbon-bound protons of amino acid ligands or of the molybdenum cofactor. The sulfite oxidase high-pH signal, on the other hand, in addition to proton spinflip transitions similar to those of xanthine ox&se, shows transitions from an exchangeable, relatively strongly coupled proton. The hyperhne coupling of this proton is not resolved in the powder lineshape because of noncolinearity of A(‘H) and g, and because of the largely anisotropic nature of its coupling. The possible signiticance in relation to the catalytic mechanism of this latter finding is discussed. 8 1985 Academic press, IIX.

In recent years techniques such as ENDOR have provided much useful information on the nature of weakly coupled nuclei (cf. 1-3) in metalloenzymes. However, simultaneous transitions of electron and nuclear spins can also be observed by ordinary EPR (4-6). These are the so-called spin-flip transitions. For nuclei that exhibit a hyperfine coupling to the electron spin there is a small probability that the system can absorb energy corresponding to the sum and the difference of the electronic and the nuclear Zeeman energies. When the nuclear Zeeman energy is large with respect to the hyperfine coupling, these transitions can be observed as satellites on either side of the main transitions, separated from the latter by the nuclear Zeeman energy (4). Under nonsaturating conditions the intensity of spin-flip transitions is small compared to that of the main (AMI = 0) lines. However spin-flip satellites can be easily observed at higher microwave powers due to the fact that they saturate much less readily than do the main lines (6). Because of the large size of its magnetic moment the nucleus for which spin-flip lines are most commonly observed is the proton (‘H). While proton spin-flip lines of free radical systems are well known (4-7), observations of similar transitions for transition-metal ions are restricted to single-crystal studies of Cu(I1) complexes (8). There appears to be no reported study of such transitions from biological systems. We report herein a study of the proton spin-flip transitions of the MO(V) EPR signals from the molybdoenzymes xanthine oxidase and sulfite oxidase. MATERIALS

AND METHODS

EPR spectroscopy. EPR spectra were recorded on a Varian E9 instrument, linked to a Digital PDP 11/lO computer and visual display unit (9). For simulation and 0022-2364185

%3.00

Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

384

PROTON

SPIN-FLIP

TRANSITIONS

385

manipulation, spectra were transferred to the University of Sussex Digital VAX 1 l/ 780 computer via a link with the PDP 1 l/10. Simulations of EPR powder lineshapes were performed by fitting to the S = f spin Hamiltonian: 27’ = PB*g.s + hS.A.1 - /3,gnB.I PI This was done using a modified version of the program QPOW written by R. L. Belford and co-workers (10-12). The nuclear g value (g,,) for ‘H was taken as 5.5856 and in all simulations the effects of the 25% naturally abundant (I = 3) isotopes 95Mo and 97Mo were ignored. Microwave power saturation profiles were fitted by iteratively minimising the sumof-squares error between calculated and experimental points, using the equation of Beinert and Orme-Johnson (13). s = k(P(1 + P/P&6)“*

PI

where S is first-derivative signal intensity, k is a constant of proportionality, P is the microwave power, PII is the power for half saturation, and b is the inhomogeneity parameter. Materials. Xanthine oxidase was prepared using the salicylate denaturation method of Hart et al. (14, 25), sulfite oxidase by the method of Lamy et al. (16), and aldehyde oxidase as described by Bray et al. (17). In general, samples were prepared in 3 mm i.d. quartz tubes, according to the procedures in references given under Results and Discussion. For xanthine oxidase samples, care had to be taken that no reduced Fe/S I center was present simultaneously with MO(V). This is because complications of the powder lineshape due to magnetic interactions between the two spin systems (18) caused difficulty in the interpretation of the EPR spectra. For some of the signals (i.e., “desulfo inhibited” and “inhibited”) the properties of the molybdenum center are such that samples could easily be prepared with only MO(V) present. Other signals had to be prepared using one-electron-reduced enzyme, in which both reduced Fe/S and MO(V) will be present, but in separate molecules. RESULTS AND DISCUSSION

Xanthine Oxidase

Xanthine oxidase is a molybdenum-iron sulfur flavoprotein that is capable of hydroxylating a wide range of compounds, including purine bases such as xanthine and many different aldehydes (19). The active site for these reactions is known to be the molybdenum (19, 20). Molybdenum in xanthine oxidase can exist in MO(N), MO(V), or Mo(V1) oxidation levels. In resting (oxidized) enzyme the molybdenum is present as Mo(VI), and on reaction with a substrate molecule this becomes reduced to Mo(IV). The electrons thus passed from substrate to enzyme can be distributed among the other redox active sites of the molecule (i.e., two [2Fe-2S] clusters and FAD) according to their relative redox potentials (21) so that a total of six electrons can be accepted. For intermediate oxidation levels (between 0- and 6-electron-reduced enzyme) an appreciable fraction of paramagnetic MO(V) is present.

386

GRAHAM Freq

330

335 Field

340 1n mT.

= 9.3490

345

NEIL GEORGE Frea

GHz

328

329 Field

330

= 9.3290

GH

331

In mT.

FIG. 1. Effect of microwave power on the powder lineshape of the xanthine oxidase 2-oxo-6-methylpurine MO(V) “very rapid” signal (9). (A) The full powder lineshape under conditions that are nonsaturatmg (upper trace, 0.01 mW) and saturating (lower trace, 400 mW) for the main AM, = 0 lines. Running conditions were temperature 49 K, and modulation amplitude 0.1 mT. (B) Detail of the g, feature, with conditions as for (A) with microwave power -, 0.01 mW, ---, 20 mW; and . - *, 400 mW. Signal intensities for all traces were normalized for clarity.

Xanthine oxidase gives rise to a large number of different MO(V) EPR signals including signals representative of intermediates of turnover in which substrate is covalently bound to molybdenum, i.e., the so-called “very rapid signal” (9). Figure 1 shows the appearance of the xanthine oxidase Ma(V) very rapid signal under nonsaturating and saturating microwave power. It can be seen that the powder lineshape changes dramatically with increasing microwave power due to the development of satellites which saturate much less readily than do the main lines (Fig. 2). The separation (Fig. 1) of these satellites from the main lines is 13.9 MHz (0.49 mT) which can be compared with the calculated proton NMR frequency at this field of 14.0 MHz (0.495 mT). This clearly indicates that the satellite lines are due to sympathetic spin inversion of a proton with the electron spin (proton spin-flip lines). Figure 3 shows an experiment similar to Fig. 1 with the xanthine oxidase “desulfo inhibited” signal. This signal results from chemical modification of the molybdenum site of the desulfo form of the enzyme with ethanediol. Studies using 13C enriched ethanediol(22) have shown that the ethanediol is bound to molybdenum in the signalgiving species. Presumably as a result of additional hyperfme coupling from the protons of the ethanediol, the desulfo inhibited signal shows particularly well-resolved proton spin-flip lines. On spectra at the highest microwave powers double proton spin-flip lines can also be seen (cf. Fig. 3e). These transitions arise from simultaneous spin flips of two protons spins and the electron spin, and their presence indicates (not surprisingly) that weak coupling to more than one proton is present. Although less well resolved, shoulders on the spin-flip lines of the xanthine oxidase very rapid signal (cf. Fig. 1B) indicate the presence of double spin-flip transitions in this species as well. The proton spin-flip lines from the desulfo inhibited signal of the related enzyme

PROTON

SPIN-FLIP

TRANSITIONS

387

POWER, mW FIG. 2. Saturation profiles for the main (AM, = 0) lines and for the proton spin-flip (AM, = +I) lines of the g, feature on the “very rapid” signal. V shows the intensity of the main lines and A that of the proton spin-flip lines (multiplied by a factor of 5 on the vertical scale) for a range of microwave powers. The lines represent the result of fitting the experimental data to Eq. [2] as described in the text. The solid lines running through V and A represent one- and two-component fits, respectively, while the broken line represents a one-component fit to the spin-flip lines (A). Signal intensity is expressed relative to the largest signal observed. The intensities of the lines were measured using computer difference to isolate the AM, = 0 and AM, = + 1 features, thus avoiding problems due to overlap of these components.

aldehyde oxidase (not illustrated) were also examined, and showed very similar behavior to those of the analogous signal from xanthine ox&se. All the xanthine oxidase signals examined gave well-resolved spin-flip lines on saturation of the main (AM, = 0) lines. Signals examined were very rapid (Fig. I), desulfo inhibited (Fig. 3), slow, inhibited, mercurial (29, and rapid (see Ref. (20) for descriptions of the slow, inhibited, and rapid signals). In all these cases except for the slow and rapid signals, development of the signals in ‘Hz0 gave no change in the intensity of the proton spin-flip lines, indicating that the protons are nonexchangeable. For the slow and rapid signals the presence of exchangeable protons with well-resolved coupling (20) caused sufficient broadening due to unresolved *H (I = 1) hyperfine structure on exchanging into *Hz0 to prevent observation of proton spin-flip lines. Possible nature of the coupled protons. Iterative computer fitting of the microwave power saturation profiles of the xanthine oxidase very rapid signal in Fig. 2 to Eq. [2] indicated that spin-flip lines from more than one type of proton were present (the profile could not be fitted with only one type of proton). An adequate fit was found with two species in the ratio 1:6 with PII2 = 244 mW and PI,* = 5.17 mW, respectively; this can be compared with Pl12 of 0.8 mW for the AM1 = 0 transitions. The value of b was found to be 1.5 in all cases. The fact that only two species were required to fit the data is probably not significant and probably only reflects a minimum number of proton types. In agreement with this, preliminary ENDOR studies (24) have indicated that between 6 and 8 nonexchangeable weakly coupled protons are present in the MO(V) rapid

388

GRAHAM

1 334

I

335

I 336

NEIL GEORGE

I 337 Field

I 338 in mT.

I 33!hi-+i-

FIG. 3. Effect of microwave power on the powder lineshape of the “desulfo inhibited’ signal. Frequency = 9.3 170 GH2. Traces (a)-(e) were run with 0.0 1, 2, 20, 50, and 400 mW microwave powers, respectively. Other running conditions were as in Fig. 1 except that the temperature was 19.3 K. A clear progression of saturation of the main lines can be seen, until in (e) the intensity of these is so small as to be negligible. The arrows in (e) indicate the position of further satellites due to double spin-flip transitions. Traces (f) show simulations of (e) for one coupled proton with colinear ( * . . ) and noncolinear (---) g and A(‘H). The (arbitrary) parameters chosen were AlI = 1.0 MHz, Al = -0.5 MHz, and Euler angles Q = 43”, @ = 27”, and y = 0” for the noncolinear simulation, using the g values given in Ref. (20)’ It should be noted that the values for the hyperfine coupling are arbitrary (no attempt was made to fit the amplitude because more than one proton is involved) and serve only to illustrate that g and A(‘H) are noncolinear.

species. The nonexchangeable nature of the protons indicates that they are most probably bound to carbon, and, in addition, for the very rapid signal, argues against any significant contribution from protons on the covalently bound substrate, as the nearest of these, the N-9 proton, is exchangeable. Thus the most likely origin of the coupled protons is either from amino acid side-chain ligands to molybdenum or from the molybdenum cofactor. The latter is a low molecular weight pterin-containing species that is associated with molybdenum in all molybdoenzymes except nitrogenase. EPR (19,20) and EXAFS (25) studies of the enzyme indicate that the environment around molybdenum is sulfur rich and this has been taken as indicating the presence of cysteinyl ligands to molybdenum (20, 25). An approximate estimation of the distance of the weakly coupled protons can be made as discussed below. In general, observation of spin-flip transitions indicates that the hyperline coupling

PROTON

SPIN-FLIP

389

TRANSITIONS

of the nucleus giving rise to the spin-flip transition must be highly anisotropic. In the case of geometrically distant nuclei the coupling will be mostly dipolar and thus this criterion will be satisfied. Under nonsaturating microwave powers the intensity of a proton spin-flip transition (I,) relative to the (unresolved) AMI = 0 lines (lo) is given approximately by (2)

where ri is the distance of the ith proton from the nucleus of the atom containing the unpaired electron (molybdenum) and Bi is the angle between the vector joining the proton and molybdenum nuclei and the magnetic field. For randomly oriented protons this reduces to [41

where reff is

given

by r,ff = [(c

l/rT)]-1/6

[51

for n equivalent protons reff = r/rP. For the very rapid signal the ratio of intensities (ZI/2Zo) was estimated by extrapolation of the saturation profiles in Fig. 2 to nonsaturating powers. Assuming that eight protons are present and that they correspond to a random orientation about g, this gives r = 3.18 .& Because of the arbitrary nature of the above assumption the margin of error in this value is probably large; nevertheless, it indicates that the protons are at a distance expected for cysteinyl carbon bound protons. SulJite Oxidase Sulfite oxidase is a molybdohemoprotein that catalyzes, at the molybdenum site, the biologically important oxidation of sulfite to sulfate. The active enzyme gives two main types of MO(V) EPR signal (Z6), the high-pH and the low-pH signals. The lowpH signal is characterized by the presence of well-resolved coupling to a single exchangeable proton (A, = 27.3 MHz) which is absent in the high-pH species. The two species can be interconverted by varying pH and chloride concentration (26) (high chloride and low pH favoring the low-pH species). Bray and co-workers (26) have proposed that the interconversion between the two species is of the form /OH Enzyme - MO = 0 + H+ + Cl- = Enzyme - MO (High-pH species)

(Law-pH species) 1~1

PI

where the OH group of the low-pH species is that bearing the strongly coupled proton. These workers also noted that the gl feature of the high-pH signal always showed small “shoulders” which they attributed to small amounts of an additional species with resolved proton coupling.

GRAHAM

L-II 332

334

336

NEIL GEORGE

338

340

342

344

Field in mT. FIG. 4. Coupling of an exchangeable proton in the sulfite oxidase high-pH signal. Frequency = 9.3140 GHz. (a) The powder lineshape in ‘H20, (b) the corresponding simulation of (a); (c) the lineshape in ‘H20, with the spin-flip lines in the g, region (which are not present in a) arrowed, (d) a simulation of(c), using the parameters in Table 1; and (e) an alternative and obviously unsatisfactory simulation with the same parameters as (d) except that g and A(‘H) are held colinear (a = fi = y = 0). The microwave power for (a) and (c) was 0.01 mW, and the temperature for all traces was 49 K. Trace (f) shows the effect of partly saturating the main transitions (power 10 mW) for the sample of trace (c) (‘H20) and (g) is the spin-flip powder lineshape for the exchangeable proton, obtained by subtracting (c) from (f). Traces (h) are simulations of(g) using noncolinear (-) and colinear (---) g and A(‘H), using the parameters in Table 1.

The sulfite oxidase high-pH signal is shown in Fig. 4, in both ‘Hz0 and 2H20 solution. The shoulders observed by Bray and co-workers can clearly be seen in the ‘H20 spectra, in Figs. 4c (arrowed) and f; they have the correct separation from the main lines for proton spin-flip transitions and have a saturation behavior typical of these. When the signal was produced in 2H20, in agreement with earlier work (26) the satellites showed a marked reduction in intensity; however, the use of high powers indicated the presence of very much weaker proton spin-flip lines (not illustrated) similar to those of xanthine oxidase; these nonexchangeable protons will not be considered further. The shoulders of Fig. 4c are therefore due to proton spin-flip transitions from a coupled exchangeable proton. The intensity of these exchangeable proton spinflip lines under nonsaturating microwave powers is large, in fact so large as to be expected to originate from a proton with a well-resolved hyperfme splitting. Two alternative explanations for this apparent discrepancy are possible. In either

PROTON

SPIN-FLIP

391

TRANSITIONS

0.05

0.w

FIG. 5. Calculated variation of hyperfine splitting and of transition probability with orientation of B for the simulation in Fig. 4d. The calculated variation of the transition probability of the AM, = 0 resonances (. . .) and the AM, = f 1 resonances (---), together with the hype&me splitting (-) is shown against the azimuthimal angle 0, the polar angle Q being fixed at an arbitrary angle of 15’. The polar coordinates are expressed relative to the diagonal frame of g (3, 2, 1 corresponding to X, y, z).

case the coupling must be essentially anisotropic (i.e., the isotropic component must be small). The first explanation to be considered is that the exchangeable proton might be rotating rapidly with respect to the molybdenum. This would cause motional averaging of the anisotropic hyperfine coupling, and thus the absence of a resolved hyperfine splitting. This possibility is considered unlikely, since it seems improbable that the degree of freedom of motion of the proton with respect to molybdenum could be so large as to cause complete motional averaging of the anisotropic hyperfine coupling. In addition the use of low temperatures (7 K) to freeze out such dynamic effects (not illustrated) gave no significant change in EPR lineshape relative to that at 49 K and thus this possibility can be excluded. The second explanation for the lack of resolved hyperfine splitting lines is noncolinearity of g with A(‘H). Figure 4 shows the appearance of the sulfite oxidase highpH signal under a variety of conditions. Figure 4g shows the spin-flip powder lineshape of the exchangeable proton obtained by subtracting trace (c) from the high-power trace (f) (10 mW). Inspection of the high-power *Hz0 spectrum (10 mW) (not illustrated) indicated that the contribution to Fig. 4g from nonexchangeable protons is minimal. With the assumption of completely anisotropic coupling (i.e., A,, = UI’), simulation (Fig. 4h) of the lineshape of Fig. 4g indicates that g and A(‘H) must be highly noncolinear. In addition, the angles of noncolinearity used to simulate the spin-flip line’ 11and I refer, respectively, to parallel and perpendicular to the vector joining the molybdenum nucleus and the proton.

392

GRAHAM

NEIL GEORGE TABLE 1

Parameters of Sulfite Oxidase MO(V) High- and Low-pH EPR Signals Value Species

Parameter

Low pH i (‘H) (MHz) High pH : (‘H) (MHz)’ Euler angles a, P, Pb

1

2

3

Reference

2.0037 24

1.9720 22

1.9658 36

12

1.9871 -10

I .9636 -10

1.9529 20

Present work Present work

45”

55”

0”

Present work

12

u It should be noted that due to the method of estimation (see results and discussion) these values are much less accurate than those obtained by direct measurement of hyperfme splitting (as for the low-pH species). b The usual convention of Rose (27) is used.

shape, together with the anisotropic nature of the coupling, make the hyperfme splitting of the main transitions small along the principal axes of g. This is demonstrated graphically in Fig. 5, which shows the calculated angular variations of the hyperfine splitting (using the parameters in Table I), together with that for the transition probabilities of the AMI = 1 and AMI = 0 resonances. It can be seen from the figure that the hyperfme splitting is close to zero near the principal axes of g (e.g., when 8 = 0), additionally the intensity of the spin-flip lines is greatest at these orientations. Thus, although a quite large proton ligand hyperhne coupling is present, noncolinearity of g with A(‘H) and the small isotropic component of A(‘H), combine to make the coupling difficult to detect. A simulation of the full powder lineshape (Fig. 4c) is shown in Fig. 4d, which can be compared with the simulation in Fig. 4e which gives the expected powder lineshape obtained with g and A(‘H) held colinear. The parameters of the sulfite oxidase high- and low-pH species are summarized in table 1. Thus it seems that for sulfite oxidase both the high- and low-pH forms of the enzyme bear a strongly coupled exchangeable proton. The strength of the coupling of the exchangeable proton in the high-pH form, together with its exchangeability, argues strongly for it being an MO-OH group, as hypothesized for the low-pH species. This raises doubts as to whether the ionizable group whose protonation influences the conversion between high- and low-pH forms, is in fact a ligand of molybdenum, as has been assumed by all previous workers (26,28), cf. Eq. [6]. An alternative hypothesis would be that the group is an amino acid side chain, or part of the molybdenum cofactor, that is close, but not detectably coupled, to MO(V), and that, like the situation for xanthine oxidase, any exchangeable protons at the molybdenum site have a pK, outside the observable range. A similar hypothesis has recently been suggested for the

PROTON

SPIN-FLIP

High pH species

\ /O-H ---MO /\

X

TRANSITIONS

393

Lou pH species

c,y H+ -

\

)‘+I

;Mo \ Cl XH+

FIG. 6. Proposed mechanism for the interconversion of the high- and low-pH Ma(V) species of sulfite oxidase. A possible mechanism for the interconversion of the high- and low-pH Ma(V) species of sulfite oxidase is shown. Group X has a pK, of about 7 (26), and is part of the molybdenum cofactor or an amino acid side chain.

interconversion of the similar high- and low-pH MO(V) species of the related enzyme Escherichia coli nitrate reductase (29). The new hypothesis is illustrated diagrammatically in Fig. 6. It should be noted that it is thermodynamically indistinguishable from that of Bray and co-workers (26) and is thus fully consistent with their data. However, these conclusions should be of very considerable importance in understanding the nature of the catalytic mechanism of the enzyme. ACKNOWLEDGMENTS The author thanks Dr. R. C. spectrum, for helpful discussion, also Ruth Williams for technical and EPR facilities were provided

Bray for drawing his attention to the “shoulders” on the sulfite oxidase and for preparations of this enzyme (prepared by Teresa Wilkinson), and assistance. The work was supported by a grant from the SERC to R.C.B. by the MRC. REFERENCES

1. B. M. HOFFMANN, J. E. ROBERTS,AND W. H. ORMEJOHNSON, J. Am. Chem. Sot. 104,860 (1982). 2. B. M. HOFFMANN, R. A. VENTERS, J. E. ROBERTS,M. NELSON, AND W. H. ORME-JOHNSON,J. Am. Chem. Sot. 104,47 11 (1982). 3. C. P. SCHOLER, A. LAGIDOT, R. MASCAREUHAS,T. INUBUSHI, R. ISAACSON,AND G. FEHER, J. Am. Chem. Sot. 104,2724 (1982). 4. H. ZELDES AND R. LIVINGSTON, Phys. Rev. 96, 1702 (1954). 5. G. T. TRAMMEL, H. ZELDES, AND R. LIVINGSTON, Phys. Rev. 110 (1958). 6. H. SHIMIZU, J. Chem. Phys. 42,3603 (1965). 7. S. SCHLICK AND L. KEVAN, J. Magn. Reson. 22, 171 (1976). 8. R. S. EACHUS, F. G. HERRING, AND Bo-LONG POH, J. Chem. Sot. A, 614 (197 I). 9. R. C. BRAY AND G. N. GEORGE, Biochem. Sot. Trans. 13,560 (1985). 10. M. J. NILCES, Ph.D. thesis, University of Illinois, 1979. 11. R. L. BELFORD AND M. J. NIU;ES, Computer Simulation of Powder Spectra, EPR Symposium, 21st Rocky Mountain Conference, Denver, Colo., 1979. 12. A. M. MAURICE, Ph.D. thesis, University of Illinois, 1980. 13. H. BEINERTAND W. H. ORME-JOHNSON,in “Magnetic Resonance in Biological Systems” (A. Ehrenber& B. G. MaImstrom, and T. V&m&d, Eds.), p. 221, Pergammon, Oxford, 1967. 14. L. I. HART, M. A. MCGARTOLL, H. R. CHAPMAN, AND R. C. BRAY, Biochem. J. 116,851 (1970). IS. R. C. BRAY, in “Flavins and Flavoproteins” (V. Massey and Williams, C. H., Eds.), p. 775, 1982. Elsevier, Amsterdam, N.Y. 16. M. T. LAMY, S. GUTTERIDGE, AND R. C. BRAY, Eiochem. .I. 185, 397 (1980).

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17. R. C. BRAY, G. N. GEORGE, S. GUTTERIDGE, L. NORLANDER, J. G. P. STELL, AND C. STUBLEY, B&hem. J. 203,263 (1982). 18. D. J. Low AND R. C. BRAY, Biochem. J. 169,471 (1978). 19. R. C. BRAY, in “The Enzymes,” 3rd ed., (P. D. Boyer, Ed.), Vol. 12, p. 299, Academic Press, New York, 1975. 20. R. C. BRAY, in “Advances in Enzymology and Related Areas of Molecular Biology” (A. Meister, Vol. 51, p. 107, Wiley, New York, 1980. 21. J. S. OLSON, D. P. BALLOU, G. PALMER, AND V. MASSEY, J. Biol. Chem. 249,4362 (1974). 22. G. N. GEORGE, D. Phil. thesis, University of Sussex, 1983. 23. G. N. GEORGE AND R. C. BRAY, Biochemistry 22,5443 (1983). 24. R. C. BRAY AND A. EHRENBERG, Personal communication. 25. S. P. CRAMER, R. WAHL, AND K. V. RAJAGOPALAN, .I. Am. Chem. Sot. 103,772l (198 1). 26. R. C. BRAY, S. GUTTERIDGE, M. T. LAMY, AND T. WILKINSON, Biochem. J. 211,227 (1983). 27. M. E. ROSE, “Elementary Theory of Angular Momentum,” Wiley, New York, 1968. 28. M. J. COHEN, I. FRIDOVICH, AND K. V. RAJAGOPALAN, J. Biol. Chem. 246,374 (1971). 29. G. N. GEORGE, R. C. BRAY, F. F. MORPETH, AND D. H. BOXER, Biochem. J. 227,925 (1985).

Ed.),