Characterization of Two 2[4Fe4S] Ferredoxins from Clostridium acetobutylicum
- 524 Downloads
In vivo hydrogen production in Clostridium acetobutylicum involves electron transfer between ferredoxin and [FeFe]-hydrogenase. Five C. acetobutylicum open reading frames were annotated as coding for putative ferredoxins. We focused our biophysical and biochemical investigations on CAC0303 and CAC3527, which possess the sequence signature and length of classical 2[4Fe4S] clostridial ferredoxins but differ significantly in theoretical pI. After cloning, heterologous expression in E. coli followed by in vitro Fe-S incorporation and purification, CAC0303 was shown to have a regular electron paramagnetic resonance (EPR) signal for a classical 2[4Fe4S] clostridial ferredoxin, while CAC3527 displayed an unusual EPR signal and a quite low reduction potential. Both ferredoxins were reduced in vitro by C. acetobutylicum [FeFe]-hydrogenase, but the CAC3527 reduction rate was 10-fold lower than that of CAC0303. These results are consistent with the efficiency of intermolecular electron transfer being dictated by the redox thermodynamics, the contribution of the ferredoxin global charge being only minor. The physiological function of CAC3527 is discussed.
KeywordsElectron Paramagnetic Resonance Electron Paramagnetic Resonance Spectrum Electron Paramagnetic Resonance Signal Methyl Viologen FeFe
O.G. was the recipient of a doctoral fellowship from the Centre National de la Recherche Scientifique and the Agence de l’Environnement et de la Maîtrise de l’Energie.
- 6.Duff JLC, Breton JLJ, Butt JN, et al. (1996) Novel redox chemistry of [3Fe-4S] clusters: electrochemical characterization of the all-Fe(II) form of the [3Fe-4S] cluster generated reversibly in various proteins and its spectroscopic investigation in Sulfolobus acidocaldarius ferredoxin. J Am Chem Soc 118:8593–8603 CrossRefGoogle Scholar
- 15.More C, Camensuli P, Dole F, et al. (1996) A new approach for the structural study of metalloproteins: the quantitative analysis of intercenter magnetic interactions. J Biol Inorg Chem 2:152–161Google Scholar
- 20.Soni BK, Soucaille P, Goma G (1987) Continuous acetone butanol fermentation: influence of vitamins on the metabolic activity of Clostridium acetobutylicum. Appl Microbiol Biotechnol 2:1–5Google Scholar