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 changed systematically to their uncharged but stereochemically equivalent carboxamide analogues. Such changes provide molecules which vary in overall charge and its surface distribution but vary minimally in structure and reduction potential. Glu-17 and Asp-6, -27, -33, -35, and -39 were converted providing six single mutants, four double mutants and one triple mutant. The proteins were characterised by UV-visible spectroscopy, square-wave voltammetry and 1H 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 k2 for the oxidation of reduced mutant ferredoxins by [Co(NH3)6]3+ were measured at 25 degrees C and I = 0.1 M by stopped-flow techniques. Each mutant displayed saturation kinetics with k2 being 30-100% of that for the recombinant protein. The rates were moderately sensitive to ionic strength. Variation in association constant K could not be detected within the confidence limits of the data. Overall the effects of the mutations were minor. In contrast to human and Anabaena 7120 [Fe2S2]-ferredoxins, electron transfer does not appear to rely on the presence of one or two specific surface carboxylate residues. It may occur from multiple sites on the surface of CpFd with recognition processes for its many physiological redox partners being controlled by relative reduction potentials, in addition to unidentified criteria. The conclusions are consistent with previous results for another series of mutant CpFd proteins interacting with physiological redox partners pyruvate: Fd oxidoreductase and hydrogenase (J.M. Moulis, V. Davasse (1995) Biochemistry 34, 16781-16788).
Brereton
P S
PS
School of Chemistry, University of Melbourne, Parkville, Victoria, Australia.
Maher
M J
MJ
Tregloan
P A
PA
Wedd
A G
AG
D003001:Cloning, Molecular; D003013:Clostridium / Q000378:metabolism*; D003035:Cobalt / Q000737:chemistry; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D013499:Surface Properties