| In living organisms, the majority of energy needed to maintain life is derived from the respiratory chain. In humans, respiration is strictly aerobic and is catalyzed by four enzyme complexes located at the inner mitochondrial membrane. In simpler prokaryotes, these complexes are either absent or substituted by other respiratory enzymes to allow them to thrive aerobically or anaerobically in diverse environments. The different respiratory chain enzymes contain a number of different prosthetic groups that form an electron transfer relay and are connected by the membrane-soluble quinone species. Using the Escherichia coli dimethyl sulfoxide reductase and succinate dehydrogenase enzymes as model systems, we set out to examine some underlying structure-function relationships that are common to respiratory chain enzymes in general. In this thesis, some questions regarding redox enzymes which we wanted to address include the elements that govern the biophysical properties of redox cofactors, the rate of electron transfer, and the mechanisms of proton transfer and quinone/quinol reduction/oxidation. This was accomplished by constructing site-directed mutants in the electron transfer subunits of dimethyl sulfoxide reductase and succinate dehydrogenase, followed by evaluations of enzyme function using in vivo complementation and in vitro biochemical, enzymology and spectroscopic techniques. |
