Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond

Abstract

The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine–thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I–IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.

Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron–Nuclear Double Resonance Spectroscopy

“…The enhanced H-bonding to the proximal cysteine in the F429H P450 2B4 causes a positive shift in the redox potential (E m ) and slows the rate of auto oxidation of the oxyferrous complex ∼ 40-fold. 19 A similar effect on E m from enhanced H-bonding interaction with the proximal cysteinyl was seen in several NOS enzymes. 14, 54 This was computationally explained by a diminished Fe-S covalency induced by strengthened H-bonding to the cysteinyl sulfur.…”

“…Since the crystal structure reveals that the mutation does not significantly perturb the structure of the active site, it suggests that water, like oxygen and CO, may bind more tightly to the iron 12,19 . As shown previously, in the presence of BP, the high-spin form of the WT enzyme essentially vanishes upon cooling to temperatures of 77 K, a change that is assigned to a repositioning of substrate in the heme pocket so that H 2 O binds as a sixth ligand.…”

“…O 2 was obtained from Matheson Co. Wild type cytochrome 2B4 and F429H were expressed in E. coli and purified to homogeneity as described 19, 29, 30 and stored at −70°C in 100 mM K phosphate buffer (pH 7.5) containing 20% glycerol. 4-(4-chlorophenyl) imidazole (CPI) was purchased from Oakwood Products, Inc., West Columbia, South Carolina.…”

“…20 The substrate free redox potential changes little with the mutation: for the F429H mutant it is −245 mV versus −300 mV for the wild type. 19 A parameter that does (inversely) correlate with H-bonding strength to the thiolate is the trans-axial Fe- CO stretching frequency. Here, the addition of the H-bond to axial thiolate in the F429H mutant shifts the frequency for the trans-axial ν Fe-C and ν C-O linkage to a higher frequency, which is indicative of diminished donation by the proximal thiolate ligand.…”

“…28 Functionally, the F429H mutation was shown to substantially slow the rate of autooxidation of oxy ferrous P4502B4 complex, and to decrease the activity by over 90%. 19 …”