40 Biochhnica etBiophysicaActa, 784 (1984)40-47 Elsevier BBA31809 SPECTROSCOPIC CHARACTERIZATION OF FERREDOXINS I AND II FROM DESULFO VIBRIO AFRICA NUS E. CLAUDE HATCHIKIAN a, RICHARD CAMMACK b.,, DAULAT S. PATIL b, A. EDWARD ROBINSON c, ANDREW J.M. RICHARDS e, SIMON GEORGE c and ANDREW J. THOMSON c a Laboratoire de Chimie Bacterienne, CNRS, B.P. 71 13277 Marseille, Cedex 9 (France), b King's College, Department of Plant Sciences, London SE24 9JF, and c School of Chemical Sciences, Unioersity of East Anglia, Norwich, NR4 7TJ (U.K.) (Received July 13th, 1983) Key words: Ferredoxin," ESR," Magnetic circular dichroism,. (D. africanus) Ferredoxins I and II isolated from Desuifccibrio africanus strain Benghazi (NCIB 8401) were examined by electron spin resonance (ESR) and magnetic circular dichroism (MCD) spectroscopy. The ferredoxins were essentially ESR-silent in the oxidized state. The spectra of the reduced ferredoxins indicated the presence of single [4Fe-4S] clusters, with intensities of approx. 1 spin per protein subunit. The spectra of reduced ferredoxin I showed two components which were interpreted as due to different aggregated forms of the protein. The midpoint reduction potentials, determined by the mediator-titration technique monitored by ESR spectroscopy, were estimated to be -385 _ 15 mV for both proteins. Thee MCD spectra of the oxidized ferredoxins confirmed that the ferredoxins were essentially diamagnetic, and were typical of [4Fe-4S] clusters in the 2 + oxidation level. MCD magnetization curves of the reduced ferredoxin I, constructed from all the major features of the MCD spectra, consistently gave intercept values of 0.5 __. 0.01 corresponding to a paramagnet with a g value near to 2.0 and a spin of S = ]. This is taken as confirmation that the proteins are exceptionally free of oxidized paramagnetic species which interfere with the spectra of other [4Fe-4SI 1+ :2 + ferredoxins. Several species of sulphate-reducing bacteria have now been found to contain more than one type of ferredoxin [1-6]. A previous paper [5] has reported the isolation and some properties of two distinct ferredoxins, termed ferredoxin I and fer- redoxin II, from the sulphate-reducing bacterium Desulfovibrio africanus. Both proteins appear to be dimers of a basic subunit containing one [4Fe-4S] cluster. Absorption spectra of the two ferredoxins differ from those of ferredoxins of other Desulfo- vibrio species by exhibiting a pronounced absorp- tion peak at 283 nm, consistent with an unusually high content of aromatic residues, tyrosine and * To whom correspondence should be addressed. Abbreviations: Hopes, 4-(2-hydroxyethyl)-l-piperazineethane- sulphonic acid; Hipip, high-potential iron-sulphur protein. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V. phenylalanine. The amino acid sequence of the subunit of ferredoxin I has recently been de- termined [7], giving a molecular weight of 6750. Ferredoxin I and ferredoxin II, which contain only four cysteine residues [5,7], represent the most simple case of single [4Fe-4S] cluster ferredoxins. Besides these two typical [4Fe-4S] ferredoxins, a complex ferredoxin designated ferredoxin III and containing more than one iron-sulphur duster per basic subunit has recently been isolated from cul- tures grown on excess iron [6]. In this paper, we present results obtained by the use of ESR spectroscopy on the iron-sulphur clus- ters in ferredoxins I and II of D. africanus. Potentiometric titrations in the presence of oxida- tion-reduction mediators, followed by ESR mea- surements were used to determine the midpoint