Two Reduced Nicotinamide Adenine Dinucleotide Dehydrogenases from the Photosynthetic Bacterium, Rhodospirillum rubrum

Abstract

Abstract Two kinds of NADH dehydrogenase, NADH:heme protein oxidoreductase (Enzyme I) (mol wt, 33,000) and NADH:quinone oxidoreductase (Enzyme II) (mol wt, 26,000) have been prepared in a homogeneous state from light-grown Rhodospirillum rubrum and characterized. Both flavin-free proteins are activated by flavins. In the presence of added flavin, Enzyme I catalyzes the reduction by NADH of cytochrome cc', cytochrome c2, quinones (ubiquinone-10, etc.), and oxidation-reduction dyes such as 2,6-dichloroindophenol at approximately identical rates, whereas II catalyzes the reduction of quinones and dyes much faster than the reduction of cytochromes. For Enzyme I, the maximum rates attained with the flavins riboflavin, flavin mononucleotide, and flavin adenine dinucleotide are in the ratio 2:1.5:1, respectively, with Km ≃ 1 µm for all three flavins. In the presence of the latter two flavins, a 4-fold excess of FAD over FMN is sufficient to depress completely the reaction rate to that observed with FAD alone. For Enzyme II, the maximum rate with FMN is twice that with FAD or riboflavin, with Km = 0.3 µm for FMN. Enzyme II has greater affinity for FMN than for FAD, because the reaction rate of the FMN-activated enzyme is not decreased by addition of as much as 10-fold excess FAD. The oxidation of NADH by oxygen catalyzed by Enzyme I is completely inhibited by ferricytochrome c2, which is preferentially reduced. At alkaline pH, cytochrome cc' stimulates the oxidation of NADH by oxygen. R. rubrum chromatophores contain two kinds of bound cytochrome-560; one is reduced by NADH, the other is reduced by succinate. By depletion of the chromatophores of Enzyme I, the extent of reduction of the first cytochrome by NADH is markedly depressed. The reaction is restored to the maximal level when purified Enzyme I is added, which suggests that the enzyme is functional in the reduction of the NADH-reducible cytochrome via a flavin associated with the chromatophores.