Protein-Protein Interactions in the Mammalian Heme Degradation Pathway: Heme Oxygenase-2, Cytochrome P450 Reductase and Biliverdin Reductase.

Protein-protein interactions involving heme oxygenase;cytochrome P450 reductase and biliverdin reductase;electron transfer complex;Biological Chemistry;Molecular;Cellular and Developmental Biology;Science (General);Science;Cellular and Molecular Biology

Spencer, Andrea L. M.Fuller, Robert S. ;

University of Michigan

关键词: Protein-protein interactions involving heme oxygenase;    cytochrome P450 reductase and biliverdin reductase;    electron transfer complex;    Biological Chemistry;    Molecular;    Cellular and Developmental Biology;    Science (General);    Science;    Cellular and Molecular Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/110320/almor_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, CO and Fe, and requires electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. The HO pathway is important for many cellular processes, including the maintenance of heme levels below toxic concentrations, iron homeostasis, carbon monoxide production, and antioxidant protection. Understanding protein-protein interactions of the heme degradation pathway will provide insight into potential regulatory mechanisms for the pathway. While the more well-studied HO isoform, HO-1, is reported to form complexes with CPR and BVR, little is known regarding the ability of HO-2 to bind either protein.In this thesis I describe various in vitro studies aimed at evaluating interactions of soluble HO-2 with CPR and BVR. 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 this residue 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 observed by cross-linking. Fluorescence quenching experiments suggest 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. Studies were also performed to evaluate the role of cysteine residues in catalytic activity and substrate inhibition for human BVR. Alanine substitution of C74 reduced catalytic activity by ~80% and this variant is less sensitive to substrate inhibition. Alanine substitution of C281, C292, C293 or C292/C293 had little effect on either catalytic activity or substrate inhibition.The studies described herein elucidate mechanisms which may be important features of regulation for the HO system in the cell, and thus have implications for regulating the cellular processes affected by HO and BVR.