The NMR spectra of naturally occurring iron-sulfur proteins have been studied for some 15 years by now1"4 and yet are not fully understood because there is disagreement between the reported spectra and those expected on a theoretical basis. We have investigated the NMR spectra of a reduced 2Fe-2S protein over a quite extended spectral width and detected some more signals that may provide the key for the assignment of the NMR spectra of every kind of ironsulfur cluster. Six to eight signals had been detected1•4"10 *in several reduced 2Fe-2S ferredoxins (one iron(III) and one iron(II)) in the range from 45 ppm downfield to 5 ppm upfield from DSS, four of them showing an anti-Curie type temperature dependence and the remaining a Curie type temperature dependence. The first idea was to assign the isotropically shifted signals to /3-CH2's of the four bound cysteines.1 7•6 Subsequently, the four signals showing anti-Curie behavior were assigned to the ß-CH2's of the cysteines coordinated to the iron(II) center.4•5

Bovine erythrocyte superoxide dismutase (SOD) in which the native zinc(I1) is substituted by cobalt(I1) has been investigated through 'H NMR spectroscopy. Owing to the magnetic-exchange coupling between cobalt(I1) and copper(II), proton signals of the histidine residues coordinated to both cobalt(I1) and copper(I1) have been observed. The signals are relatively narrow, Le., of the same quality as those of the copper-deprived cobalt(I1) SOD. Assignment of histidine NH signals is obtained through deuterium exchange, and a tentative assignment of the other signals is proposed on the basis of T I and T2 measurements. Anions like N3-, NCO-, and NCS-cause large variations in the position of the histidine proton signals. The same kind of variations, although to a smaller extent, occur at pH 9, probably due to the binding of the OH-anion. The spectra have been interpreted in terms of one histidine (possibly His 44) being removed from coordination by the anion ligand. The electronic and CD spectra of Cu2C02SOD and its derivatives as compared to those of native SOD derivatives indicate that the two systems behave in a quite similar fashion.Bovine erythrocyte superoxide dismutase (SOD hereafter) is an enzyme containing zinc(I1) and copper(I1) ions, the latter in the site at which the catalytic reaction occurs.' the US. Army, through its European Research Office (Contract

a model 1280 computer and operated at frequencies of 361.062 MHz for proton and 90.793 MHz for I3C observation. The pulse sequence employed for the experiment was essentially that of Freeman2' modified to deliver a composite 180' pulse.40 Pulse widths were 30 ps for carbon and 32 ps for proton Lia the decoupler coils. Fixed delays around the acquisition pulse (Al and Az) were set to 3.0 and 2.5 ms (respectively) with phase cycling to provide the equivalent of quadrature data in both dimensions. The initial S(tl,tz) data matrix was generated using 256 x 2K blocks of data followed by processing in the usual fashion. The experiment was performed on a sample prepared by dissolving 200 mg of 1 in approximately 2 mL of deuteriochloroform. Accumulation of the initial s(t,Jz) data matrix required approximately 3 h. The contour plot erence spectrum was the projected sum of the data matrix through the F, dimensionj6 and the carbon reference spectrum was the projection through the F2 dimension.Acknowledgment. This paper is dedicated to Professor William von Eggers Doering on the occasion of his 65th birthday. His excitement about the science of chemistry has been continuously transmitted to M.R.W. for the past 25 years. small way, this enthusiasm has also been transmitted to all of the other authors M.R.W. wish to thank the Robert A.with a spectral width of *833 Hz for proton and *3425 Hz for carbon, Of this paper. Of the authors, G.E.M., M.A., A.J'W3, and (Figure 1) was prepared by "sing four contour levels. The proton refand for the establishment and operation of the South Carolina (40) Levitt, M. H.; Freeman, R.Abstract: The pH dependence of the 'H NMR spectra of active-site specifically substituted cobalt(I1) horse liver alcohol dehydrogenase and its complexes with NAD' and NADH are reported. The 'H NMR signals of the cysteine and the histidine ligands are well shifted from the diamagnetic position. The 6-NH of histidine 67 probably deprotonates with a pKa of 9.0 f 0.2; in the complex with NAD' the same group exhibits a pH-dependent shift without deprotonation with a pK, of 8.3 f 0.2. The complex with NADH is pH independent up to pH 9.3. Both the 'H NMR and near-IR spectra indicate that no major change in the coordination sphere of the catalytic metal ion occurs upon binding coenzyme. The results suggest the participation in catalysis of groups not considered in previously proposed mechanisms.

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