Insights into the alkaline transformation of ferricytochrome c from (1)H NMR studies in 30% acetonitrile-water.

Recently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous-nonaqueous media, resulting in the formation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One-dimensional (1D) and two-dimensional (2D) (1)H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline-like forms in 30% acetonitrile-water solution. A native-like (M80-ligated) III* form, two lysine-ligated forms (IVa* and IVb*), and a hydroxide-ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D (1)H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20 degrees C and 35 degrees C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for the hydroxide-ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.

Resonance Raman spectroscopy of soybean peroxidase.

Resonance Raman spectra (600-1700 cm-1) for the heme enzyme soybean peroxidase (Rz = 2.5) were obtained using Soret band excitation at 406.7 nm. The vibrational frequencies and depolarization data indicate a strong similarity between the active sites of soybean and horseradish peroxidase. This similarity suggests that the active site in the resting form of soybean peroxidase contains a ferric iron, is a high-spin 5-coordinate heme binding His as a fifth axial ligand.

Preparation, purification, and spectrophotometric characterization of cytochrome P450 1A2 conjugated with polyethylene glycol monomethyl ether.

Rabbit cytochrome P450 1A2 was modified with succinimidyl carbonate poly(ethylene glycol) monomethyl ether, purified by size exclusion high performance liquid chromatography, and lyophilized. Modification of cytochrome P450 1A2 caused no structural deformation of the heme as evidenced by the similarity of the spectral signatures for both the ferric form and the ferrous-CO complex to the respective forms for the unmodified enzyme. Ethoxyresorufin O-deethylation activity in the presence of iodosobenzene for the modified enzyme was comparable to that of the native enzyme.

Direct electrochemistry for the imidazole complex of microperoxidase-11 in dimethyl sulfoxide solution at naked electrode substrates including glassy carbon, gold, and platinum.

The effect of the total water content on the persistence and rate of direct heterogeneous electron transfer between the imidazole complex of microperoxidase-11 (im-MP-11) and naked gold, platinum, and glassy carbon (GC) in dimethyl sulfoxide (DMSO) solutions containing 0.1 M tetra-n-butylammonium perchlorate was investigated using cyclic voltammetry. Electron transfer between im-MP-11 and Au, Pt, and GC has been found to be persistent for more than 1 h and at least quasi-reversible [k(s)' = (8.7 ± 0.1) × 10(-4) cm/s (Au), k(s)' = (7.2 ± 1.3) × 10(-4) cm/s (Pt), and k(s)' = (5.7 ± 1.0) × 10(-4) cm/s (GC)] in dimethyl sulfoxide containing an absolute water content between 0.1 and 1.8%. The heterogeneous electron transfer rate constant is independent of the total water content of the DMSO solution when between 0.1 and 1.8% water is present.

Effect of pegylation on the structure and function of horse cytochrome c.

The preparation and spectrophotometric characterization of (both Fe2+ and Fe3+ forms) poly(ethylene glycol) (PEG; av FW 5000)-modified horse cytochrome c (cyt c(PEG)n) with different degrees of modification (nav = 6, 19) by UV-vis spectroscopy, circular dichroism spectroscopy, resonance Raman spectroscopy, and cyclic voltammetry are described. Extensive modification (nav = 19) of cyt c causes gross structural deformation of the heme as evidenced by major spectral changes in the UV-vis and circular dichroism spectral signatures of both the Fe2+ and Fe3+ forms. Modification of cyt c by six PEG residues, however, produces a protein in which the heme active site is structurally and functionally intact (UV-vis, circular dichroism, and resonance Raman) and which exhibits at least quasireversible direct electron transfer (Eo' = 338 +/- 5 mV vs SHE; (2.1 +/- 0.6) x 10(-3) cm/s) at bis(4-pyridyl) disulfide-modified Au electrodes.