A comparison of the initial rate kinetics for human biliverdin-IX␣ reductase and biliverdin-IX␤ reductase with a series of synthetic biliverdins with propionate side chains "moving" from a bridging position across the central methene bridge (␣ isomers) to a "␥-configuration" reveals characteristic behavior that allows us to propose distinct models for the two active sites. For human biliverdin-IX␣ 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␣ reductase, albeit poor ones. In the case of mesobiliverdin-XIII␣, 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␣ reductase, we suggest that a pair of positively charged side chains play a key role in optimally binding the IX␣ isomers. In the case of human biliverdin-IX␤ 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␤ reductase is potently inhibited by mesobiliverdin-XIII␣ and protohemin, which is consistent with the hypothesis that the tetrapyrrole and flavin substrate bind at a common site.

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