Cytochrome P-450 contains a cysteine thiolate ligand at the axial position of (porphinato)iron(III) core. The axial thiolate has been shown to promote the heterolysis of the coordinated dioxygen upon sequential donation of electron and proton. 1-3 The crystallographic structure of cytochrome P-450 cam reported by Poulos et al. 4 has suggested the presence of weak double NH‚‚‚S hydrogen bonds between S of Cys 357 and two amide NHs of Gly 359 and Gln 360, as shown in Figure 1a. Recently, the presence of two clear NH‚‚‚S hydrogen bonds was reported for the crystal structure of chloroperoxidase. 5 Similar hydrogen bonds are found in the active site of P-450 BM-3 , 6 P-450 terp , 7 and P-450 eryF . 8 For P-450 cam , a molecular dynamics simulation from the reported crystallographic structure results in the shortening of the NH‚‚‚S hydrogen bond distances. 9 Furthermore, comparison of the Protein Data Bank crystallographical structural parameters at the active sites of substrate-bound P-450 cam with substrate-free P-450 cam 10 indicates shorter N‚‚‚S distance at the active site of the substrate-bound P-450 cam accompanied by a positive shift of the reported redox potential. 11 A remarkable effect on the electrochemical properties by NH‚‚‚S hydrogen bond have already been revealed by us for various metal thiolate complexes 12-14 as well as iron-sulfur proteins. [15][16][17] In order

Iron(III) porphinate complexes of arenethiolate having single or double NH···S hydrogen bonds at the axial position, [FeIII(OEP){S-2,6-(RCONH)2C6H3}] (R = CF3 (1), CH3 (2)) or [FeIII(OEP)(S-2-RCONHC6H4)] (R = CF3 (3), CH3 (4), t-Bu (5)), were synthesized as models of P-450 and chloroperoxidase. The presence of an NH···S hydrogen bond in these complexes is confirmed by their crystal structures in the solid state, the IR shift of the amide NH band and the direct through-bond contact-shift of the amide 2H NMR signal in benzene. The NH···S hydrogen bond elongates the Fe−S bond distance, stabilizes the Fe(III) state, protects the complexes from decomposition by air and moisture, and shifts the redox to more positive potentials. These functions by the hydrogen bond are more significant than the effect of steric hindrance.

The primary sequence of Cys-X-Gly-Y- (X, hydrophobic residue; Y, hydrophilic residue) is highly conserved in cytochrome P-450s. The amide NHs of Leu, Gly, and X are assumed to form NH.S hydrogen bonds which are also found in the active site fragment, Cys-Pro-Ala-Leu, of chloroperoxidase (CPO). [Fe(III)(OEP)(Z-cys-Leu-Gly-Leu-OMe)] (OEP, octaethylporphinato; Z, benzyloxycarbonyl) and [Fe(III)(OEP)(Z-cys-Pro-Ala-Leu-OMe)] were synthesized as P-450 and CPO model complexes containing the invariant amino acid fragment of the active site. The corresponding gallium(III) complexes were also synthesized to investigate the solution structures using two-dimensional (2D) NMR experiments because the Ga(III) ion is similar to the Fe(III) ion in the ionic radii and in the coordination geometry. The solution structures of the peptide part of the gallium complexes indicate that the invariant fragments maintain a beta I-turn-like conformation and then form NH.S hydrogen bonds between S(gamma)Cys and NH of the third and fourth amino acid residues. The hydrogen bonds have also been confirmed by the (2)H NMR spectra of N(2)H-substituted Fe(III) peptide complexes. The Fe(III)/Fe(II) redox potentials of the Fe(III) complexes indicate that the NH.S hydrogen bonds in the fragments causes a slight positive shift of the redox potential. The tri- and tetrapeptide Fe(III) complexes containing the invariant fragments of P-450 are kinetically stable at 30 degrees C in CH(2)Cl(2). In contrast, [Fe(III)(OEP)(Z-cys-Leu-OMe)] decomposed to give [Fe(II)(OEP)] (22%) and the corresponding disulfide immediately in CD(2)Cl(2) at 30 degrees C for 1 h. These results indicate that the invariant fragments involving the hydrogen bonds cause the stabilization of the high-spin Fe(III) resting state rather than the positive shift of Fe(III)/Fe(II) redox potential.

Gallium(III)-substituted P-450 models, [Ga(OEP)(SAr)] (OEP=octaethylporphinato; SAr=S-2-CF3CONHC6H4, SPh), were synthesized and the structures were determined by X-ray analysis. The gallium(III) complexes were found to be excellent structural analogues having a diamagnetic metal center.