Initial-rate kinetics of the flavin reductase reaction catalysed by human biliverdin-IX beta reductase (BVR-B)

The initial-rate kinetics of the flavin reductase reaction catalysed by bil iverdin-IX beta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH bindi ng to the free enzyme was characterized using stopped-flow fluorescence que nching, and a K-d of 15.8 mu M was calculated. Equilibrium fluorescence que nching experiments indicated a K-d of 0.55 mu M, suggesting that an enzyme- NADPH encounter complex (K-d 15.8 mu M) isomerizes to a more stable 'nucleo tide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K-m values of 52 mu M, 125 mu M and 53 mu M, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K-i of 76 mu M, indicating that interactions with the isoallox azine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase act ivities of the enzyme exhibit a sharp optimum at pH 5 in citrate buffer. An initial-rate study indicated that the enzyme obeys a steady-state ordered mechanism in this buffer. The initial-rate kinetics in sodium acetate at pH 5 are consistent with a rapid-equilibrium ordered mechanism, indicating th at citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverd in XIII alpha, a synthetic biliverdin which binds to flavin reductase but d oes not act as a substrate for the enzyme, exhibits competitive kinetics wi th FMN (K-i 0.59 mu M) and mixed-inhibition kinetics with NADPH. This is co nsistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IX beta reductase has also been shown to exhibit ferric reductase activity, w ith an apparent K-m of 2.5 mu M for the ferric iron. The ferric reductase r eaction requires NAD(P)H and FMN. This activity is intriguing, as haem clea vage in the foetus produces non-alpha. isomers of biliverdin and ferric iro n, both of which are substrates for flavin reductase/biliverdin-IX beta red uctase.