Regulation of the Reduced Nicotinamide Adenine Dinucleotide Phosphate-Ferredoxin Reductase System in Clostridium kluyveri

Abstract The mechanism of regulation of NADPH-ferredoxin reductase was studied in cell-free lysates of Clostridium kluyveri. The following activities, which are assumed to be linked to the enzyme, were investigated: ferredoxin reduction by NADPH, NADP+ reduction by reduced ferredoxin, transhydrogenation from NADPH to NAD+, and methyl viologen reduction by NADPH. Ferredoxin reduction by NADPH is controlled by the oxidation-reduction state of the NAD+-NADH couple. NAD+ is an obligatory activator (Michaelis activation constant, Ka = 0.9 x 10-4 m), which increases Vmax, while the Km of the substrate NADPH (Km = 2.25 x 10-5 m) remains unaffected. β-NAD+ analogues can substitute to varying degrees for β-NAD+, while α-NAD+ and NMN or AMP analogues are totally inactive. NADH is an inhibitor, competitive to NAD+ rather than to NADPH. NADP+ reduction by reduced ferredoxin is subject to product inhibition; NADPH is competitive to NADP+ (Km = 1.52 x 10-4 m). The transhydrogenation from NADPH to NAD+ is stimulated by oxidized and inhibited by reduced ferredoxin; ferredoxin is not involved in the electron flow. Methyl viologen reduction by NADPH is not controlled by either NAD+ or NADH.