Energy Coupling in Cytochrome c Oxidase

University Of South Florida, Tampa FL

Investigators

Abstract

The ability of heme/copper terminal oxidases to transduce energy is the fulcrum of cellular respiration in aerobic organisms. In fact, evolution of life on earth relies on the ability of aerobic organisms to metabolize oxygen. Heme/copper oxidases are found in nearly all aerobic organisms including some archaeobacteria, some prokaryotes, and nearly all eukaryotes. Despite the importance of these enzymes to our understanding of the evolution of aerobic life on earth, a detailed understanding of enzymatic function has not yet been achieved. The overall goal of this project is to identify the magnitudes and time scales of molar volume and enthalpy changes associated with each electron transfer reaction of the enzymes catalytic cycle using variable temperature/pressure transient absorption spectroscopy and photothermal methods. Specifically, electrons will be photo-injected into fully oxidized (no active proton transport) cytochrome c oxidases (CcO) from both bovine heart tissue and the bacteria Rb. sphaeroides using Ru(II)-based photoinitiators. Volume and enthalpy profiles associated with the subsequent intramolecular ET will be constructed using photothermal spectroscopy and variable temperature/pressure transient optical spectroscopy. These experiments will be repeated using CcO containing both P- and F-intermediates localized at the binuclear center (i.e., electron transfer believed to be coupled to active proton transport). Successful completion of this project will provide the first look into the conformational dynamics and free-energy coupling associated with active proton transport in these complex enzymes. Broader Impact: The research plan provides multi-disciplinary educational opportunities for students, who will receive broad training in modern experimental physical chemistry including photothermal techniques and fast optical spectroscopy as well as biochemical methods including protein chemistry.