|All aerobic life forms rely on a membrane bound respiratory chain that ends in an enzyme capable of reducing O2 to H2O. This enzyme, an oxidase, also performs energy transduction to convert redox free energy into a transmembrane charge gradient. In mammals the respiratory chain is located in the inner mitochondrial membrane, whereas in aerobic bacteria it is an integral part of the plasma membrane. The current paradigm is that the terminal enzymes from these diverse aerobic respiratory chains are related structurally, and are all members of a cytochrome oxidase family.
I am interested in describing the molecular mechanism of O2 reduction, and in the details of energy transduction. Since these enzymes employ the transition metals iron and copper during catalysis a variety of spectroscopic approaches are used to characterize the status of these functional groups and their surroundings. We have used transient-state optical spectroscopy to define elementary steps in O2 reduction, and this approach is being extended to study the transmembrane reactions involved in energy transduction. We have also been funded recently for an electron paramagnetic resonance spectrometer that will be used to characterize further the transition metals and to begin looking at the role of protein-based free radicals as intermediates in these reactions. We have for the most part used purified cytochrome c oxidase from beef heart muscle because of the abundant protein available from this source, and we have begun to study the respiratory chain of the strict aerobe Bacillus subtilis.
The B. subtilis system allows us to apply the powerful methods of molecular biology. We have isolated two oxidases from B. subtilis that have a high degree of homology with mitochondrial cytochrome oxidase. We have made gene-disrupted strains for each of these enzymes and are now undertaking site-directed mutagenesis. During the course of this work we have found a homolog of a protein first found in yeast (Sco1) that is required in the assembly of the copper centres of cytochrome c oxidase. We have cloned the gene for this protein from B. subtilis, known as YpmQ. We have made a strain in which YpmQ is deleted from the chromosome of B. subtilis and this strain is unable to produce functional cytochrome c oxidase. We can complement this knock out by expression of YpmQ from a plasmid. We will use this plasmid-based expression system to isolate native and mutant forms of YpmQ. These will be characterized for their metal binding properties and used to study metal transfer to apo-cytochrome c oxidase.