Prof. Ólöf Einarsdóttir - UCSC Biomedical Research Faculty

Figure from Ludwig et al. Chembiochem. 2001, 2,392-403

Survival of most cells, tissues and organisms depends on their ability to extract energy from food using dioxygen. In eukaryotic organisms, this process of respiration takes place in mitochondria. The final member in this respiratory chain is cytochrome oxidase, which reduces dioxygen to water and plays a key role in regulating the rate of mitochondrial respiration and ATP production.

The bovine heart cytochrome oxidase consists of thirteen different subunits. Mitochondrial genes code the three largest subunits, two of which contain four redox active metal centers (heme a₃, Cu_B, heme a, and Cu_A), while the remaining subunits are the products of nuclear genes.

Einarsdóttir's laboratory focuses on elucidating the mechanism of electron and proton transfer during the reduction of dioxygen to water by heme-copper oxidases, such as cytochrome oxidase. Using time-resolved spectroscopic techniques, Einarsdóttir and her colleagues have been able to probe the temporal evolution of the enzyme's protein structure during turnover conditions.

One of the laboratory's three primary goals is analysis of dioxygen reduction to water by heme-copper oxidases on nanosecond and longer time scales. To accomplish this, they use a CO-flow-flash method, in which the dioxygen reduction is initiated by photodissociation of the CO complex in the presence of O₂. They also examine the reaction of oxygen with the unliganded, reduced enzyme, using dioxygen that is produced in situ by photodissociating synthetic dioxygen complexes, such as dicobalt µ-peroxo polyamine complexes. Time-resolved multichannel optical absorption spectroscopy, in conjunction with singular value decomposition (SVD) and global exponential fitting analysis, is used to follow the kinetics of electron and proton transfer and to deduce the UV-Vis spectra of the transient intermediates. Time-resolved magnetic circular dichroism (TRMCD) spectroscopy allows them to probe the electronic structure of the hemes in the various intermediates, while FTIR spectroscopy is used to investigate conformational changes that occur during electron and proton transfer reactions.

A comprehensive and experimentally validated model for the intramolecular electron transfer pathways in the heme-copper oxidases is not yet available. Because bacterial oxidases are subject to greater experimental control and are less complex than the bovine cytochrome oxidase, Einarsdóttir's laboratory is currently investigating intramolecular electron transfer in the bacterial oxidases. For this work, they use a photo-activatable dye, thiouredopyrene-trisulfonate (TUPS), covalently linked to single reactive cysteine residues on the oxidases. Time-resolved optical absorption spectroscopy, in conjunction with SVD and global exponential fitting, is being used to determine the spectra of the present intermediates, as well as the rate constants of individual electron transfer steps. By mutating appropriately located residues in the cysteine-free bacterial oxidase, varying the distance between the labeled cysteine and the initial electron acceptor, and by introducing breaks into presumed electron transfer pathways by site-directed mutagenesis, they hope to obtain detailed information regarding intramolecular electron transfer pathways in heme-copper oxidases.

Recent X-ray crystallographic studies have shown that the active site of bovine heart cytochrome oxidase contains a tyrosine residue (Tyr244), cross-linked to histidine 240, which is a ligand to Cu_B at the active site of the enzyme. This tyrosine is ideally situated to participate in dioxygen activation and O-O bond cleavage by acting as a hydrogen donor. Einarsdóttir's laboratory, in collaboration with UCSC Professor Joe Konopelski's research group, is currently making chemical analogs of the active site of cytochrome oxidase, including the His-Tyr cross-linked dipeptide and the cyclic pentapeptide (His-Pro-Glu-Val-Tyr), with and without Cu-ligands incorporated. These analogs are then studied using a multi-spectroscopic approach, including steady-state and time-resolved UV-Vis spectroscopy, FTIR and ESR.

The approaches and results described above have already significantly broadened our understanding of fundamental energy conservation pathways involving cytochrome oxidase. Cytochrome oxidase reduces dioxygen to water through a series of highly reactive oxygen intermediates. If the enzyme is not properly assembled or is catalytically abnormal, these reactive oxygen species (ROS) can be released from the active site. This leads to oxidative stress, which has been associated with aging and certain degenerative diseases. For instance, defects in energy metabolism contribute to the pathogenesis of Alzheimer disease. It has been reported that cytochrome oxidase in late-onset Alzheimer patients shows anomalous kinetic behavior and decreased activity in brain and peripheral tissue. Such abnormal forms of the enzyme are caused by mutations in mitochondrial cytochrome oxidase genes, resulting in increased production of ROS in the brain. Recent studies have also suggested that the b-amyloid peptide, the core component of plaques present in Alzheimer patients, causes a significant reduction in mitochondrial respiration by inhibiting cytochrome oxidase.

As their work progresses, Einarsdóttir and her colleagues expect to provide new insights into the relation between cytochrome oxidase function, ROS and oxidative stress, which will be valuable to both biomedical research and potential clinical applications.