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

Stable multilayer electroactive films were grown on rough pyrolytic graphite (PG) electrodes by alternate adsorption of layers of polyanion poly(styrenesulfonate) (PSS) and positively charged myoglobin (Mb) from their aqueous solutions. Incorporation of large amounts of electroactive Mb was facilitated by high electrode surface areas and by adsorbing coiled PSS from 0.5 M NaCl solutions. Cyclic voltammetry of {PSS/Mb}n films showed a pair of well-defined, chemically reversible peaks at about -0.25 V vs SCE at pH 5.5, characteristic of the Mb heme Fe III /Fe II redox couple. Electroactivity was extended to 7 {PSS/Mb} bilayers on rough PG surfaces, in comparison to 2 electroactive layers on smooth gold coated with mercaptopropanesulfonic acid. Square wave voltammograms of {PSS/Mb}n films gave good fits by nonlinear regression analysis to a model featuring dispersion of formal potentials, providing average formal potentials and an apparent rate constant. Oxygen and trichloroacetic acid were catalytically reduced by Mb in {PSS/Mb}n films with significant decreases in the electrode potential required. Making such films on PG rather than on gold provides a larger number of electroactive layers with no need for chemical pretreatment of the electrode.

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Stable protein-clay films were fabricated by casting an aqueous dispersion of protein and clay on pyrolytic graphite electrodes. Myoglobin (Mb), hemoglobin (Hb), and horseradish peroxidase (HRP) in clay films gave a pair of well defined, quasi-reversible cyclic voltammetric peaks at about -0.28 V vs SCE in pH 5.5 buffers, characteristic of the protein heme Fe III /Fe II redox couples. Square wave voltammograms (SWV) of the protein-clay films gave good fits by nonlinear regression analysis to a model that featured thin-layer SWV and formal potential dispersion, providing average apparent heterogeneous electrontransfer rate constants, ks, and average formal potentials, E°′. UV-vis and reflectance absorption infrared spectra showed that the proteins in clay films retained near-native secondary structures. X-ray diffraction revealed that Mb-clay and Hb-clay films feature ordered layered structures with Mb and Hb intercalated between clay layers. Incorporated HRP induced disorder in the clay films. Oxygen, trichloroacetic acid, nitrite, and hydrogen peroxide were catalytically reduced by all three proteins in clay films.