title: Protein/protein interactions in the mammalian heme degradation pathway: heme oxygenase-2, cytochrome P450 reductase, and biliverdin reductase.
abstract: Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, 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 precede formation of the productive electron transfer complex. The (1)H-(15)N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, 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 Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in K(m) values 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 (K(d) = 15.1 μM). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the (1)H-(15)N 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 free heme (dissociated from HO) on BVR fluorescence.
authors.affiliation: From the Cellular and Molecular Biology Training Program and.
authors.forename: Andrea L M
authors.initials: AL
authors.lastname: Spencer
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48103 and.
authors.forename: Ireena
authors.initials: I
authors.lastname: Bagai
authors.affiliation: the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48103 and zuiderwe@umich.edu.
authors.forename: Erik R P
authors.initials: ER
authors.lastname: Zuiderweg
authors.affiliation: From the Cellular and Molecular Biology Training Program and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48103 and sragsdal@umich.edu.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D001059:Apoproteins / Q000378:metabolism; D002850:Chromatography, Gel; D003374:Coumarins / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000378:metabolism; D005453:Fluorescence; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D016296:Mutagenesis / Q000235:genetics; D050505:Mutant Proteins / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
chemical_list: D001059:Apoproteins; D003374:Coumarins; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D006418:Heme; C030123:coumarin; D003903:Deuterium; D006419:Heme Oxygenase (Decyclizing); C105245:heme oxygenase-2; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Cytochrome P450; Electron Transfer Complex; Enzyme Kinetics; Enzyme Mechanism; Heme Oxygenase; Nuclear Magnetic Resonance (NMR); Protein/Protein Interaction