Isolation and Some Properties of NAD⁺ Reductase of the Green Photosynthetic Bacterium Prosthecochloris aestuarii

Abstract

NAD⁺ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration.
This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder), 452, 411, and 385nm (the 411nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119, 000. The enzyme catalyzes the reduction of NAD⁺ and NADP⁺ by photoreduced spinach ferredoxin or reduced benzyl viologen. It also catalyzes the reduction of cytochromes and dyes such as benzyl viologen and 2, 6-dichloroindophenol (DCIP) with NADH or NADPH as the electron donor. The reduction of cytochrome c-555(550) of this organism is accelerated by the addition of cofactors such as menadione. In these reactions, the enzyme is more specific for NAD+ or NADH than for NADP⁺ or NADPH. Reduction of cytochrome c or dyes catalyzed by the enzyme is strongly inhibited by rotenone or amytal, but not by antimycin A or o-phenanthroline.
It is suggested that NAD⁺ photoreduction in this organism takes place via direct electron flow with the mediation of ferredoxin and flavoprotein enzyme, as in Chlorobium and green plants.