Voltammetry and visible and infrared spectroscopy were used to explore protonation equilibria coupled to electron transfer between electrodes and the heme protein myoglobin (Mb) in thin liquid crystal films of didodecyldimethylammonium bromide (DDAB) and phosphatidylcholines (PC). Mb conformation and heme iron ligation in the films were controlled by the pH of the external solution. Acid-base equilibrium models successfully explained pH dependencies of Soret band absorbances, formal potentials, electron transfer rate constants, and electroactive surface concentrations of Mb in the films. A pK a1 of 4.6 in the Mb-lipid films is associated with protonation of histidine residues in hydrophobic regions of the Mb structure, possibly involving the proximal histidine bound to iron and/or the distal histidine in the heme pocket. At pH < 4.6, a partly unfolded molten globule form of Mb predominates in the films and is reduced directly. Native metmyoglobin [MbFe(III)-H 2 O] appears to be the major species in films between pH 5.5 and 8. In this pH range, protonation of MbFe(III)-H 2 O occurs prior to electron transfer, and a protonated form which may be a kinetic conformer accepts the electron. MbFe(III)-OH is formed in the films at pH > 9, and its oneelectron reduction is also coupled to protonation. Experimental SectionMaterials and Solutions. Horse myoglobin (Sigma) dissolved in buffer was passed through Amicon YM30 filters

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In previous work, greatly enhanced rates of electron transfer were found for myoglobin (Mb) in ordered films of surfactants on pyrolytic graphite (PG) electrodes. Direct electron transfer is now reported for Mb in films of didodecyldimethylammonium bromide (DDAB) on platinum, tin-doped indium oxide, and gold electrodes. Rates of electron transfer in these films were similar on all electrodes. In the absence of surfactant, electron transfer was observed on bare electrodes only when Mb was purified by chromatography, and only on hydrophilic tin-doped In2O3 or PG. Treatment of tin-doped In2O3 or PG electrodes with unpurified protein solutions blocked electron transfer to Mb in the purified solutions. Reflectance-absorbance infrared and X-ray photoelectron spectroscopy revealed proteinaceous adsorbates on electrodes exposed to unpurified solutions of Mb. This adsorbate blocks electron transfer to Mb and to ferricyanide in solution. Results suggest that electron transfer in the Mb-DDAB films may be facilitated partly by strong adsorption of surfactants on electrodes. Surfactant adsorbed at electrode-film interfaces appears to inhibit adsorption of macromolecules from Mb solutions which could otherwise block electron transfer between Mb and electrodes.

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