Infrared detection of functional waters and protonatable amino acids; solving the proton-electron coupling mechanism of cytochrome oxidase

Cytochrome c oxidase is a component of the human energy-providing respiratory chain that is responsible for consumption most of the oxygen that we breathe. It uses products of metabolism of food to reduce the oxygen to water. Energy that is released by the reaction is used to drive protons across the membrane in which it resides. This proton gradient is used to drive formation of ATP, the major supplier of energy needed by the myriad of reactions within our cells. We already know a great deal about the atomic structure of the enzyme and the cofactors that catalyse the oxygen-consuming chemistry. What has been much more difficult to determine is how these reactions cause the proton movements that conserve the released energy. This arises because protons cannot be 'seen' in atomic models derived from X-ray studies of crystals and because the paths on which they travel are likely to be dynamic structures that change through the catalytic cycle. Mid-infrared vibrational spectroscopy (IR or 'FTIR' spectroscopy) provides the means measure changes in proteins as they change between different states by measuring tiny changes in the way that the proteins absorb infrared light. These infrared changes are sufficiently well resolved that we can interpret them in terms of specific atomic movements of groups of atoms, or of chemical changes of specific amino acids or cofactors within the protein. By causing the protein to change between two states that are important for its function, we can IR spectroscopy to 'see' atomic changes that are occurring. The technique is most powerful and useful when crystal structures have already produced a static atomic structure, as is the case for cytochrome oxidase; in such cases we can combine information from static X-ray structures with that from IR spectroscopy to provide a description of the dynamic mechanism of catalysis. It is becoming increasingly clear that water molecules buried within proteins often have roles in catalysis that are as central as those of amino acids and cofactors. Roles for water in proton transfer pathways are particularly important. We have a good idea of how the coupling of proton transfer in cytochrome oxidase works in principle, but it is probably the lack of ability to monitor functional water involvement that has precluded detailed understanding of the atomic mechanism. In recent years, it has become feasible with IR spectroscopy to directly measure such water molecules. We have carried out initial studies with cytochrome oxidase and can clearly see such structural waters molecules that change in a manner that suggests a role in proton transfer catalysis. In this project, we aim to monitor how these water molecules change in concert with specific amino acid and cofactor changes. For many enzymes, the important steps of a typical catalytic reaction occur on the microseconds-milliseconds range, as is the case for cytochrome oxidase, and it is this range of timescales that we will be measuring. The overall outcome of the work will provide a description the basic mechanism of energy conservation in cytochrome oxidase and this will help us understand how this enzyme functions and malfunctions in health and disease. It will also provide insights into the importance of water in enzyme catalysis more generally. Finally, the technology itself is unique in the UK -- it will provide a basic resource to study water in other enzyme systems and is also being developed for practical applied applications in medical diagnostics.

IR signals arising from functional water molecules in bovine mitochondrial cytochrome c oxidase (CcO) will be detected and characterised by static and time-resolved FTIR spectroscopy. IR signatures of strongly and weakly H-bonded waters and protonated water clusters have been defined and detected in other proteins, and all are predicted to play key roles in our working model of proton electron coupling. We have already collected data showing the existence of weakly and strongly H-bonded waters. Static and TR-FTIR data will be accumulated by transmission or attenuated total reflection methods, dependent on which is most suitable for the reaction under study. Transients will be initiated with a 10 mnsec, 532nm Nd/YAG laser and collected in step scan mode to 5 microsecond timeslices. Signal/noise will be maximised by averaging of transients, optimisation of sample optical parameters and the use of filters to select narrow IR spectral regions that allow the IR intensity at measured frequencies to be substantially increased. All of these methods are already implemented. Signal assignments and interpretations will be informed by our published IR data on static spectra, from direct experimental measurements and DFT calculations of normal vibrational modes (using Gaussian '03) of relevant model compounds and from effects of H/D and 18O/16O exchange. Specific transitions that will be analysed are: CO photolysis from fully reduced CcO; haem-haem electron transfer initiated by CO photolysis from 'mixed-valence' CcO; selective oxidation-reduction of metal centres; oxidation of reduced CcO with 18O2 versus 16O2. The results on water changes will be combined with IR data on protonation and other changes of amino acids and cofactors to generate a full description of how these elements combine to produce the proton/electron coupling mechanism, the pathways for proton transfer and the mechanisms by which essential gating between them is achieved.