Protein-Protein Interactions in the Mammalian Heme Degradation Pathway: Heme Oxygenase-2, Cytochrome P450 Reductase and Biliverdin Reductase [Protein Structure and Folding]

September 7th, 2014 by Spencer, A. M., Bagai, I., Becker, D. F., Zuiderweg, E. R. P., Ragsdale, S. W.

Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO and Fe with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. Here we describe mutagenesis experiments combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, crosslinking, gel filtration and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that proceed formation of the productive electron transfer complex. The 1H 15N TROSY NMR spectrum of HO-2 reveals specific residues, including L201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues L201 and K169 cause a respective 3- and 22-fold increase in Km for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2/CPR complex (Kd= 15.1 μM). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by crosslinking. For example, under conditions that CPR affects the 1H 15N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of heme (dissociated from HO) on BVR fluorescence.
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