Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant.

摘要: Abstract All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays crucial role in their mechanism of action. Conversion the Cys436 to Ser NH2-truncated microsomal CYP2B4 (ΔCYP2B4) transforms enzyme into two-electron NADPH oxidase producing H2O2 without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine effects this ligation change on spin-state and coordination structure ΔC436S CYP2B4, magnetic circular dichroism electronic absorption spectra several oxidation/ligation states variant have been measured compared with those structurally defined complexes. The substrate-free ferric mutant are indicative high-spin five-coordinate ligated by anionic serinate. spectroscopic properties dithionite-reduced (deoxyferrous) protein (high-spin) state, it is concluded has protonated yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes sixth ligands (NO, CO, O2) examined also likely serine, as would be expected for such hydroperoxo-ferric catalytic intermediate. Ligation vs. deprotonated cysteine result large difference acidity. Thus, necessary push cysteinate, although can accept two electrons protons, unable heterolytically cleave O–O bond species generate Compound I hydroxylate substrate.