Structures of Thiolate- and Carboxylate-Ligated Ferric H93G Myoglobin:  Models for Cytochrome P450 and for Oxyanion-Bound Heme Proteins^,

Abstract

Crystal structures of the ferric H93G myoglobin (Mb) cavity mutant containing either an anionic proximal thiolate sulfur donor or a carboxylate oxygen donor ligand are reported at 1.7 and 1.4 A resolution, respectively. The crystal structure and magnetic circular dichroism spectra of the H93G Mb beta-mercaptoethanol (BME) thiolate adduct reveal a high-spin, five-coordinate complex. Furthermore, the bound BME appears to have an intramolecular hydrogen bond involving the alcohol proton and the ligated thiolate sulfur, mimicking one of the three proximal N-H...S hydrogen bonds in cytochrome P450. The Fe is displaced from the porphyrin plane by 0.5 A and forms a 2.41 A Fe-S bond. The Fe(3+)-S-C angle is 111 degrees , indicative of a covalent Fe-S bond with sp(3)-hybridized sulfur. Therefore, the H93G Mb.BME complex provides an excellent protein-derived structural model for high-spin ferric P450. In particular, the Fe-S bond in high-spin ferric P450-CAM has essentially the same geometry despite the constraints imposed by covalent linkage of the cysteine to the protein backbone. This suggests that evolution led to the geometric optimization of the proximal Fe-S(cysteinate) bond in P450. The crystal structure and spectral properties of the H93G Mb acetate adduct reveal a high-spin, six-coordinate complex with proximal acetate and distal water axial ligands. The distal His-64 forms a hydrogen bond with the bound water. The Fe-acetate bonding geometry is inconsistent with an electron pair along the Fe-O bond as the Fe-O-C angle is 152 degrees and the Fe is far from the plane of the acetate. Thus, the Fe-O bonding is ionic. The H93G Mb cavity mutant has already been shown to be a versatile model system for the study of ligand binding to heme proteins; this investigation affords the first structural evidence that nonimidazole exogenous ligands bind in the proximal ligation site.

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

“…The EA spectra for the ferric species had a shift in the Soret peak position from 417 nm for the wild-type (data not shown) to 404 nm for the mutant [5]. The EA and MCD spectra of the ΔC436S CYP2B4 mutant in the ferric state are most similar to those of the five-coordinate tyrosinate-ligated ferric H93Y Mb [33] as well as the phenolate adduct of ferric H93G Mb [23](Fig. 2, A and C).…”

“…However, the high-spin charge transfer band for ferric H93(AcO − ) Mb adducts is red-shifted to 617 nm (vs. 604 nm for the phenolate complex) and the MCD spectrum of acetate-bound H93G Mb in this region is non-symmetric (with more intense trough at ~630 nm than a peak at ~605 nm). It has recently been established by crystallography that the acetate adduct of ferric H93G Mb is six-coordinate with acetate and water as ligands [23]. Based mainly on the close spectral similarity to ferric H93Y Mb (Fig.…”

“…Preparation of the H93G Mb phenolate (PhO − ) adduct was achieved by addition of phenol in dimethyl sulfoxide (DMSO) to exogenous-ligand-free ferric protein (~50 µM) [23]. After examination of the ferric state of each protein (~1.5 mL in a 1 cm septum-sealed cuvette), the samples were extensively purged over 30 min with a continuous flow of nitrogen while on ice.…”

“…The H93G myoglobin (Mb) “cavity mutant” is an especially useful system for modeling heme proteins [20,21,22]. We have compared the MCD and EA spectra of ΔC436S CYP2B4 to those of H93G Mb with different O-donor ligands, such as phenolate (PhO − ), benzoate (BzO − ) and acetate (AcO − ) [23,24], as well as to relevant states of H93Y Mb, wild-type Mb and horseradish peroxidase (HRP), in order to determine the ligation state and establish the heme iron coordination structure of this interesting P450 mutant.…”

Aerobic oxidation of β-isophorone by tetraphenylporphyrin catalysts in pyridine solution

“…The spin states for weak field ligands are high spin, which applies to pyridine as well as chloride and acetate [28,29]. The spin crossover occurs for stronger field ligands such as isothiocyanate [30,31]. The molecules were geometry optimized until a convergence of <10 −6 Ha per iterative step was reached [32].…”