Conversion of mitochondrial cytochrome b5 into a species capable of performing the efficient coupled oxidation of heme.
Histidine-63, one of the heme axial ligands in outer mitochondrial membrane cytochrome b5 (OM cyt b5) has been replaced by a methionine. The H63M variant performs the efficient and regioselective coupled oxidation of heme in order to produce >90% of the alpha-isomer of verdoheme. The variant was characterized by electronic, EPR, and NMR spectroscopic studies which indicate that the ferric form is a high-spin species whose heme is coordinated by histidine-39 in the proximal site and likely by water in the distal site. The coordination of methionine to the ferric heme was ruled out on the basis of NMR spectroscopic studies. Addition of imidazole to a solution of the ferric variant results in the formation of a species axially coordinated by imidazole and histidine-63. The reduction potential of the variant was found to be +110 mV in the absence of exogenous imidazole and -92 mV in the presence of imidazole. These values compare well with the reduction potential of myoglobin (50 mV) and wild-type OM cyt b5 (-102 mV), respectively, consistent with the axial ligation described above. The ferrous variant, on the other hand, is a low-spin species coordinated by histidine-39 and methionine-63. Carbon monoxide (CO) readily displaces Met-63 from its coordination site on the ferrous heme, whereas CO cannot completely displace Met-63 from its coordination site on verdoheme. Consequently, the mechanism of inhibition for the oxidation of verdoheme to iron-biliverdin in the H63M variant appears to be similar to that observed for the heme-heme oxygenase complex in the presence of CO.
Rodríguez
J C
JC
Department of Chemistry, Oklahoma State University, Stillwater 74078-3071, USA.
Rivera
M
M
D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008715:Methionine / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D006418:Heme; D006639:Histidine; D009249:NADP; D015786:Cytochromes b5; D008715:Methionine; D006419:Heme Oxygenase (Decyclizing)