Bilirubin Reductase from Mediterraneibacter gnavus: Positional Reduction Preferences and Transient-State Analysis

The first committed step for the excretion of bilirubin is reduction of four vinylic bonds to form urobilinogen. This reaction is catalyzed by bilirubin reductase (BilR) that uses four NADHs to accomplish the transformation. Due to the asymmetry of bilirubin, BilR must either recognize 15 different oxidant substrates or have a preferred reduction order. AlphaFold structural prediction indicates structural homology with bacterial 2,4-dienoyl-CoA reductase and 2-napthoyl-CoA reductase, both of which are single-subunit proteins with a two-domain structure that houses three cofactors: an FAD, an Fe₄S₄ cluster, and an FMN. In this study, anaerobic kinetic and mechanistic analysis of bilirubin reductase from Mediterraneibacter gnavus (MgBilR) is presented. Anaerobic solvent exchange studies define the stereochemistry of hydride transfer from NADH as ProS. Transient-state kinetics of the reductive half-reaction demonstrate that MgBilR can become reduced by four electrons with either NADH or NADPH, but that NADH is the preferred reductant by four orders of magnitude (based on k_red/K_d values). Only the first hydride transfer from NADH occurs at a rate that is catalytically relevant. Single-turnover experiments show two dominant phases corresponding to two-electron flavin reduction and subsequent rate-limiting bilirubin reduction that occurs with flavin reoxidation. Single-turnover data correlated with steady-state assays and NMR tracking of the steady-state reaction confirm that BilR can reduce both types of vinylic bonds of bilirubin but appears to have a 4-fold preference for reduction of α-vinylic over enamine bonds that is defined almost entirely by the rate of hydride transfers to the oxidant.