Mycobacterium tuberculosis FprA, a novel bacterial NADPH‐ferredoxin reductase

The gene fprA of Mycobacterium tuberculosis , encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50‐kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant ( k cat / K m ) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD‐NADPH and FADH 2 ‐NADP + ) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one‐electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe−2S] iron‐sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A K m value of 30 n m and a specificity constant of 110 µ m −1 ·s −1 (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH‐ferredoxin oxidoreductase.