Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4.
A fundamental aspect of cytochrome P450 function is the role of the strictly conserved axial cysteine ligand, replacement of which by histidine has invariably resulted in mammalian and bacterial preparations devoid of heme. Isolation of the His-436 variant of NH2-truncated P450 2B4 partly as the holoenzyme was achieved in the present study by mutagenesis of the I-helix Ala-298 residue to Glu and subsequent conversion of the axial Cys-436 to His. The expressed A298E/C436H double mutant, cloned with a hexahistidine tag, had a molecular mass equivalent to that of the primary structure of His-tagged truncated 2B4 and the sum of the two mutated residues, and contained a heme group which, when released on HPLC, showed a retention time and spectrum identical to those of iron protoporphyrin IX. The absolute spectra of A298E/C436H indicate a change in heme coordination structure from low- to high-spin, and, as expected for a His-ligated hemeprotein, the Soret maximum of the ferrous CO complex is at 422 nm. The double mutant has no oxygenase activity with representative substrates known to undergo transformation by the oxene [(FeO)3+] or peroxo activated oxygen species, but catalyzes significant H2O2 formation that is NADPH- and time-dependent, and directly proportional to the concentration of A298E/C436H in the presence of saturating reductase. Moreover, the catalytic efficiency of A298E/C436H in the H2O2-supported peroxidation of pyrogallol is more than two orders of magnitude greater than that of wild-type 2B4 or the A298E variant. The results unambiguously demonstrate that the proximal thiolate ligand is essential for substrate oxygenation by P450.
Vatsis
Kostas P
KP
Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, MI 48109-0606, USA. kvatsis@umich.edu
Peng
Hwei-Ming
HM
Coon
Minor J
MJ
D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D008024:Ligands; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013053:Spectrophotometry
D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D006861:Hydrogen Peroxide; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D003545:Cysteine