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

Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron­(III) and the hyperfine couplings of the heme-pyrrole ¹⁴N but has relatively little effect on the ¹H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe­(III)–S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe­(II) enzyme, which is trapped in the conformation of the parent Fe­(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an <i>S</i> = ³/₂ state with the anion radical exchange-coupled to the Fe­(II). In the mutant, it localizes on the iron, generating an <i>S</i> = ¹/₂ Fe­(I) state. (iii) The additional H-bond has little effect on <i>g</i> values and ¹H–¹⁴N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I

Binding of Yeast Cytochrome <i>c</i> to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome <i>c</i> Peroxidase: Isothermal Titration Calorimetry

Previously, we constructed, expressed, and purified 46 charge-reversal mutants of yeast cytochrome <i>c</i> peroxidase (CcP) and determined their electronic absorption spectra, their reaction with H₂O₂, and their steady-state catalytic properties [Pearl, N. M. et al. (2008) Biochemistry 47, 2766−2775]. Forty-four of the mutants involve the conversion of either an aspartate or glutamate residue to a lysine residue, while two are positive-to-negative mutations, R31E and K149D. In this paper, we report on a calorimetric study of the interaction of each charge-reversal mutant (excluding the internal mutants D76K and D235K) with recombinant yeast iso-1 ferricytochrome <i>c</i>(C102T) (yCc) under conditions where only one-to-one yCc/CcP complex formation is observed. Thirteen of the 44 surface-site charge-reversal mutants decrease the binding affinity for yCc by a factor of 2 or more. Eight of the 13 mutations (E32K, D33K, D34K, E35K, E118K, E201K, E290K, E291K) occur within, or on the immediate periphery, of the crystallographically defined yCc binding site [Pelletier, H. and Kraut, J. (1992) Science 258, 1748−1755], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the “back-side” of CcP. The current study is consistent with a model for yCc binding to CcP in which yCc binds predominantly near the region defined by crystallographic structure of the 1:1 yCc–CcP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex

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

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