Trp³⁵⁹ regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA

Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp³⁵⁹, proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp³⁵⁹ is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp³⁵⁹. W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with - 230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k_cat and NAD(P)H K_m values, but this led to improvements in catalytic efficiency (k_cat/K_m) with NADH as reducing coenzyme (9.6/18.8 μM^-1·min^-1 respectively, compared with 5.7 μM^-1·min^-1 for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s ^-1 for wild-type FprA and 4.8 s^-1/13.4 s^-1 for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp³⁵⁹ for flavin stability. The results demonstrate that the conserved Trp³⁵⁹ is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.