Abstract
Rhodobacter sphaeroides MDC 6521 was able to produce bio-hydrogen (H2) in anaerobic conditions under illumination. In this study the effects of the hydrogenase inhibitor—diphenylene iodonium (Ph2I) and its solvent dimethylsulphoxide (DMSO) on growth characteristics and H2 production by R. sphaeroides were investigated. The results point out the concentration dependent DMSO effect: in the presence of 10 mM DMSO H2 yield was ~6 fold lower than that of the control. The bacterium was unable to produce H2 in the presence of Ph2I. In order to examine the mediatory role of proton motive force (∆p) or the F0F1-ATPase in H2 production by R. sphaeroides, the effects of Ph2I and DMSO on ∆p and its components (membrane potential (∆ψ) and transmembrane pH gradient), and ATPase activity were determined. In these conditions ∆ψ was of –98 mV and the reversed ∆pH was +30 mV, resulting in ∆p of –68 mV. Ph2I decreased ∆ψ in concentrations of 20 μM and higher; lower concentrations of Ph2I as DMSO had no valuable effect on ∆ψ. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity sensitive to N,N′-dicyclohexylcarbodiimide. The 10–20 μM Ph2I did not affect the ATPase activity, whereas 40 μM Ph2I caused a marked inhibition (~2 fold) in ATPase activity. The obtained results provide novel evidence on the involvement of hydrogenase and the F0F1-ATPase in H2 production by R. sphaeroides. Moreover, these data indicate the role of hydrogenase and the F0F1-ATPase in ∆p generation. In addition, DMSO might increase an interaction of nitrogenase with CO2, decreasing nitrogenase activity and affecting H2 production.

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