The initial-rate kinetics of the flavin reductase reaction catalysed by biliverdin-IXbeta reductase at pH 7.5 are consistent with a rapid-equilibrium ordered mechanism, with the pyridine nucleotide binding first. NADPH binding to the free enzyme was characterized using stopped-flow fluorescence quenching, and a K(d) of 15.8 microM was calculated. Equilibrium fluorescence quenching experiments indicated a K(d) of 0.55 microM, suggesting that an enzyme-NADPH encounter complex (K(d) 15.8 microM) isomerizes to a more stable 'nucleotide-induced' conformation. The enzyme was shown to catalyse the reduction of FMN, FAD and riboflavin, with K(m) values of 52 microM, 125 microM and 53 microM, respectively. Lumichrome was shown to be a competitive inhibitor against FMN, with a K(i) of 76 microM, indicating that interactions with the isoalloxazine ring are probably sufficient for binding. During initial experiments it was observed that both the flavin reductase and biliverdin reductase activities 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 that citrate may directly affect the enzyme's behaviour at pH 5. Mesobiliverdin XIIIalpha, a synthetic biliverdin which binds to flavin reductase but does not act as a substrate for the enzyme, exhibits competitive kinetics with FMN (K(i) 0.59 microM) and mixed-inhibition kinetics with NADPH. This is consistent with a single pyridine nucleotide site and competition by FMN and biliverdin for a second site. Interestingly, flavin reductase/biliverdin-IXbeta reductase has also been shown to exhibit ferric reductase activity, with an apparent K(m) of 2.5 microM for the ferric iron. The ferric reductase reaction requires NAD(P)H and FMN. This activity is intriguing, as haem cleavage in the foetus produces non-alpha isomers of biliverdin and ferric iron, both of which are substrates for flavin reductase/biliverdin-IXbeta reductase.