The general objective of this project is to contribute to understanding of function and structure-function relationships in redox metalloproteins, primarily via observations of spectroscopy and dynamics. Our approach emphasizes vibrational (including time-resolved) spectroscopies, which are sensitive to conformations, redox states, proton transfers, etc. - in short, many of the things one needs to know to understand protein function and structure-function relationships. We also propose to use other techniques and develop new technologies as necessary to answer important scientific questions. During the upcoming grant period we will concentrate on the heme-copper oxidases, where key questions about the energy transduction processes that power aerobic life remain unanswered. We propose the following Specific Aims: Aim 1: Electron transfer. O2 reduction, and redox-linked proton transport in heme-copper oxidases: These are the key processes in aerobic bioenergetics. We will elucidate the interactive roles of the active sites, protein structure, and intraprotein water during the function of the oxidases. Specific projects include (a) infrared (IR) flow-flash studies of O2 reduction, (b) isotopic substitution and mutant studies, (c) studies of intraprotein electron transfer, and (d) studies of intraprotein water and proton transport. Aim 2: Technology development, which is required to support some of the projects proposed. This includes (a) Applications of microfluidics and IR microscopy to time-resolved IR, (b) IR spectroscopy applied to monolayer protein electrochemistry, and (c) nuclear resonance vibrational spectroscopy (NRVS) applied to heme-copper oxidases.