Investigation of the role of surface residues in the ferredoxin from Clostridium pasteurianum

Eleven mutant forms of the ferredoxin from Clostridium pasteurianum (CpFd; 2 Fe4S4; 6200 Da)have been isolated in which six surface carboxylates are c hanged systematically to their uncharged but stereochemically equivalent ca rboxamide analogues. Such changes provide molecules which vary in overall c harge and its surface distribution but vary minimally in structure and redu ction potential. Glu-17 and Asp-6, -27, -33, -35 and -39 were converted pro viding six single mutants, four double mutants and one triple mutant. The p roteins were characterised by UV-visible spectroscopy, square-wave voltamme try and H-1 NMR. Their ability to mediate electron transfer between spinach NADH:ferredoxin oxidoreductase and horse heart cytochrome c was assessed. Each mutant is 30-100% as active as the recombinant protein with the triple mutant D33,35,39N being least active. Second-order rate constants k(2) for the oxidation of reduced mutant ferredoxins by [Co(NH3)(6)](3+) were measu red at 25 degrees C and I = 0.1 M by stopped-flow techniques. Each mutant d isplayed saturation kinetics with k(2) being 30-100% of that for the recomb inant protein. The rates were moderately sensitive to ionic strength. Varia tion in association constant K could not be detected within the confidence limits of the data. Overall the effects of the mutations were minor. In con trast to human and Anabaena 7120 [Fe2S2]-ferredoxins, electron transfer doe s not appear to rely on the presence of one or two specific surface carboxy late residues. It may occur from multiple sites on the surface of CpFd with recognition processes for its many physiological redox partners being cont rolled by relative reduction potentials, in addition to unidentified criter ia. The conclusions are consistent with previous results for another series of mutant CpFd proteins interacting with physiological redox partners pyru vate:Fd oxidoreductase and hydrogenase (J.M. Moulis, V. Davasse (1995) Bioc hemistry 34, 16781-16788). (C) 1999 Elsevier Science B.V. All rights reserv ed.