Electron transfer reactions are a key process in the energy cycle of many living organisms, such as bacteria or in the eukaryotic mitochondria. The transfer of electrons is achieved by a network of metalloproteins, which couple the delivery and receipt of electrons to catalytic transformations of biological chemistry. We investigated the redox chemistry of an electron shuttle, cytochrome c₅₅₄, a tetraheme electron transfer protein from Nitrosomonas europaea and a diheme cytochrome c peroxidase from Shewanella oneidensis.

Cytochrome c₅₅₄ is an important multiheme protein that participates in the cycle of ammonia oxidation in Nitrosomonas europaea, which is an essential element of the energy metabolism of this bacterium. In our investigations we generated a platform to study the kinetics and the mechanism of the electron transfer of the protein. In addition to resolving all of the four heme potentials, we have also showed previously unobserved unidirectional electron transfer.

While cytochrome c₅₅₄ is simply an electron shuttle, the diheme bacterial peroxidase uses the electrons for the catalysis of hydrogen peroxide that protects the cells against reactive oxygen species (ROS). The initial biophysical characterization of the Shewanella oneidensis peroxidase by UV/Visible and EPR spectroscopy and steady state kinetic investigations demonstrate that the formerly unstudied Shewanella enzyme can be classified among the traditional class of peroxidases, where an activation step is required prior to catalysis.

Further kinetic investigation of the mechanism of Shewanella oneidensis CcP was carried out by rapid mixing stopped flow electronic absorption spectroscopy. Our results demonstrate the formation of two optically detectable intermediates in the mechanism. The second order rate constant for peroxide what is found to be 5.7×10⁷ M^-1s^-1, which is similar to be the only available data from the Pseudomonas aeruginosa peroxidase.

Redox coupled activation of the Shewanella peroxidase includes a loop motion near the peroxidatic active site. We have shown evidence for the conformational change in the loop by protein film voltammetry and MCD spectroscopy. The spectral assignments of the hemes are carried out and evidence of the six to five coordinate change at the active site heme is shown.