Direct electrochemistry and enzymatic activity of bacterial polyhemic cytochrome c3 incorporated in clay films

Quartz crystal microbalance and voltammetric measurements are used to study the electroenzymatic activity of incorporated bacterial polyhemic cytochrome c3 in clay minerals. Two different natural clays have been chosen, i.e., kaolinite and montmorillonite, which differ in structure, swelling property and cation exchange capacity. Comparative voltammetric studies of various electroactive species at clay-modified pyrolytic graphite electrodes are first undertaken, which allow the complete characterization of the clay deposit. Then, the incorporation process of bacterial polyhemic cytochrome c3 in the two clays is studied coupling quartz crystal microbalance and voltammetric measurements. Based on their respective characteristics, each clay yields a different behavior of the incorporated cytochrome c3, with significantly different electroactive fractions of the immobilized protein. The consequences of the interactions between the protein and the clays on the enzymatic activity of cytochrome c3 is then further examined. Firstly, the influence of the incorporation of cytochromes in clays on the metal reductase activity developed by bacterial polyheme c-type cytochromes is discussed. Secondly, hydrogenase enzymatic reactions are studied inside the clay films. An electrode is constructed by immobilizing hydrogenase in the clay and progressively incorporating either artificial (methyl viologen) or physiological (cytochrome c3) hydrogenase partner. It is shown that this modified electrode yields an efficient and stable response for either H2 consumption or evolution over a large range of pH.