An analysis of activity determinations in a series of coupled redox reactions with special reference to hydrogenase.

In this paper a spectrophotometric method is described to study the effects of pH and redox potential on the hydrogen production activity of hydrogenase, which will be applied in the following paper. The theoretical part of this paper gives the mathematical description of a system in which a redox mediator is continuously reduced by a redox system, in a non-rate-limiting way and continuously enzymatically oxidized, in a rate-limiting way. Due to the changes in redox potential during the course of the reaction the ratio reduced/oxidized redox mediator declines gradually. It is possible to relate the changes in this ratio to the actual amount of product formed. The calculation of product formation has been applied to the production of hydrogen by hydrogenase in a system with a redox mediator, whose reduced form functions as the ultimate electron donor for hydrogenase, and the dithionite/(bi)sulphite redox couple as reducing system. The changes in the ratio reduced/oxidized redox mediator as a function of pH and redox potential, followed spectrophotometrically, have been related to the hydrogen production activity of hydrogenase. The second part of the paper deals with the analysis of the theory and describes the conditions under which the method can be used with methyl viologen as redox mediator. The conditions for determining the pH dependence of the hydrogen production activity manometrically are also described.