Characterization of coenzyme binding and selectivity determinants in Mycobacterium tuberculosis flavoprotein reductase A: Analysis of Arg¹⁹⁹ and Arg²⁰⁰ mutants at the NADP(H) 2′-phosphate binding site

Mycobacterium tuberculosis FprA (flavoprotein reductase A) is an NAD(P)H- and FAD-binding reductase that is structurally/evolutionarily related to adrenodoxin reductase. Structural analysis implicates Arg¹⁹⁹ and Arg²⁰⁰ in interactions with the NADP(H) 2′-phosphate group. R199A, R200A and R199A/R200A mutants were characterized to explore the roles of these basic residues. All mutations abolished neutral FAD semiquinone stabilization in the NADPH-reduced enzyme, owing to weakened NADPH affinity. Instead, FAD hydroquinone was formed in all mutants, and each displayed substantially enhanced autooxidation rates (20-40-fold) compared with NADPH-reduced WT (wild-type) FprA. Steady-state ferricyanide reduction studies revealed diminished NADPH affinity (higher K_m values), but lower NADH K_m values. Despite a lowered k_cat, the R199A/R200A mutant exhibited a 200-fold coenzyme specificity switch towards NADH, although substrate inhibition was observed at high NADH concentrations (K_i = 250 μM). Stopped-flow FAD reduction studies confirmed substantially increased NADPH K_d values, although the limiting flavin reduction rate constant was similar in all mutants. The R199A mutation abolished electron transfer between hydroquinone FprA and NADP⁺, while this reaction progressed (via an FADH₂-NADP⁺ charge-transfer intermediate) for R200A FprA, albeit more slowly (k_lim = 58.1 s^-1 compared with > 300 s^-1) than in WT. All mutations caused positive shifts in FAD potential (∼40-65 mV). Binding of an NADPH analogue (tetrahydro-NADP) induced negative shifts in potential (∼30-40 mV) only for variants with the R200A mutation, indicating distinctive effects of Arg¹⁹⁹/Arg²⁰⁰ on coenzyme binding mode and FAD potential. Collectively, these data reveal important roles for the phylogenetically conserved arginines in controlling FprA FAD environment, thermodynamics, coenzyme selectivity and reactivity.