Biophysical investigation of bacterial cytochrome c peroxidase by protein film voltammetry and spectroscopy.

摘要

The reduction of molecular oxygen is the major source of energy within living cells. The four electron, four proton coupled reduction contains several toxic intermediates before producing H2O. These intermediates, known as reactive oxygen species (ROS), are strong oxidants. They have been shown to be oxidized proteins and damage DNA as well as enzyme co-factors. Nature has created several regulatory enzymes to protect vital cellular components from ROS.;Reactive oxygen species are also generated extracellularly as a means of self-defense. Mammalian macrophages such as granulocytes and neutrophils produce superoxide and ultimately hydrogen peroxide to defend the host cell from pathogens. But the pathogens have developed a defense mechanism of their own. Bacteria such as the gram-negative Pseudomonas aeruginosa and the pathogenic Escherichia coli contain the multi-heme enzyme cytochrome c peroxidase (CcP), which catalyzes the two-electron reduction of hydrogen peroxide to water with the aid of an endogenous electron donor. Here we describe the first investigation of Pseudomonas aeruginosa cytochrome c peroxidase using protein film voltammetry. This technique was also used to examine a new class of bacterial peroxidase from Escherichia coli.;Bacterial cytochrome c peroxidases have been studied since the 1970s beginning with the enzyme from Pseudomonas aeruginosa . Many of its properties have been determined using a variety of biophysical techniques. Pa CcP contains two heme cofactors. Each heme has a specific function within the enzyme and work together to reduce H2O2. The electrochemical technique of protein film voltammetry allowed the catalytic mechanism to be further elucidated. In our investigation insight into the rate-limiting step of catalysis was gained as well as the key features involved in activation.;Protein film voltammetry was also useful in investigating the bacterial peroxidase from Escherichia coli. The E. coli enzyme is unique as the primary sequence suggests that it contains a third heme-binding motif. Here we describe the first expression and characterization of this triheme bacterial peroxidase. Standard biophysical techniques such as electron paramagnetic resonance and solution kinetic assays were used in conjunction with protein film voltammetry to examine the properties of this new class of peroxidases.