Studies on the specificity of the tetrapyrrole substrate for human biliverdin-IXα reductase and biliverdin-IXβ reductase -: Structure-activity relationships define models for both active sites

comparison of the initial rate kinetics for human biliverdin-IX alpha reductase and biliverdin-IX beta reductase with a series of synthetic biliverdins with propionate side chains "moving" from a bridging position across the central methene bridge (alpha isomers) to a "gamma-configuration" reveals characteristic behavior that allows us to propose distinct models for the two active sites. For human biliverdin-IX alpha reductase, as previously discussed for the rat and ox enzymes, it appears that at least one "bridging propionate" is necessary for optimal binding and catalytic activity, whereas two are preferred. All other configurations studied were substrates for human biliverdin-IX alpha reductase, albeit poor ones. In the case of mesobiliverdin-XIIII alpha, extending the propionate side chains to hexanoate resulted in a significant loss of activity, whereas the butyrate derivative retained high activity. For human biliverdin-IX alpha reductase, we suggest that a pair of positively charged side chains play a key role in optimally binding the IX alpha isomers. In the case of human biliverdin-IX beta reductase, the enzyme cannot tolerate even one propionate in the bridging position, suggesting that two negatively charged residues on the enzyme surface may preclude productive binding in this case. The flavin reductase activity of biliverdin-IX beta reductase is potently inhibited by mesobiliverdin-XIII alpha and protohemin, which is consistent with the hypothesis that the tetrapyrrole and flavin substrate bind at a common site.