Direct monitoring of the electron pool effect of cytochrome c3 by highly sensitive EQCM measurements.

Cytochrome c(3) from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c(3) with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c(3) molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c(3) is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c(3) to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c(3) and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c(3) stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c(3) facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c(3) donates the accepted electrons to the viologen with difficulty.