SUMMARY: Hydrogenases from and reduced methylene blue, ferricyanide, benzyl- and methyl-viologens when hydrogen was the donor. Methylene blue was the most effective acceptor. Hughes press preparations of either organism in tris (2-amino-2-hydroxymethyl propane-1:3-diol) buffer (pH 8·0) resulted in the best extraction of the enzyme. Hydrogenase from was readily inactivated by traces of oxygen but this could be prevented by sodium dithionite. Pyridine nucleotides and cytochrome are reduced by hydrogenase, but in extracts of Azotobacter, however, the addition of iron was required to couple DPNH to cytochrome The mechanism appears to involve the reduction of Fe to Fe enzymically and the Fe is oxidized by cytochrome non-enzymically. The effect of pH value and nature of buffer on this system was examined. Mo did not reduce cytochrome , with or without hydrogenase.

Metal-deficiency experiments, inhibitor studies, activation of dialysed preparations, and the results of radioactive tracer assays of purified protein fractions, showed that iron is the main metal constituent of hydrogenase. Molybdenum, however, is required for the fixation of nitrogen.


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