Spectroscopic and electrochemical studies of horse myoglobin in dimethyl sulfoxide

Abstract.

This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and suggest that helix A has moved away from the heme. UV-vis and RR spectra for deoxyMb in DMSO suggest that the heme iron is six-coordinate low-spin, most likely coordinating DMSO. Addition of CO to deoxyMb in DMSO produces a single, photostable six-coordinate CO adduct. UV-vis and RR for Mb-CO in DMSO are consistent with a six-coordinate low-spin heme iron binding His93 weakly, if at all. The polarity of the distal heme pocket is comparable to that of the closed form of horse Mb-CO in aqueous solution, pH 7. Direct electron transfer between horse Mb and Au in DMSO solution was investigated by cyclic voltammetry. Mb exhibits stable and well-defined electrochemical responses that do not appear to be affected by the water content (1.3–7.5%). The electrochemical characteristics are consistent with a one-electron, quasi-reversible, diffusion-controlled charge transfer process at Au. E° for horse Mb in DMSO at Au is –0.241±0.005 V vs. NHE. The formal heterogeneous electron transfer rate constant, calculated from ΔE _p at 20 mV/s, is 1.7±0.5×10^–4 cm/s. The rate, which is unaffected by the presence of 1.3–7.5% water, is competitive with that previously reported for horse Mb in aqueous solution.