Multi-electron multi-proton reactions form the basis of nearly every chemical reaction involved in energy storage and manipulation. Despite their importance, the basic properties of these chemical transformations, such as the details of how electron transfer and proton-coupled redox events that must occur during these reactions are controlled, remain poorly understood. The sulfite and nitrite reductase family of enzymes are responsible for carrying out the six-electron reduction of sulfite to sulfide and nitrite to ammonia, respectively. These enzymes play fundamental roles in microbial metabolism and are either dissimilatory or assimilatory in nature. Multi-electron multi-proton reactions are investigated by the study of the catalytic mechanisms of two enzymes that are structurally different, but carry out similarly complex chemistry: the dimeric multi heme cytochrome c nitrite reductase from Shewanella oneidensis and the monomeric siroheme and [4Fe-4S] cluster containing sulfite reductase from Mycobacterium tuberculosis. Employing protein electrochemistry the properties of electron transfer steps and proton-coupled redox steps that occur throughout the catalytic cycle of cytochrome c nitrite reductase during its reduction of substrate revealed the strategies employed by this enzyme. The results presented indicate the reduction of substrate by the enzyme occurs in a series of one electron steps rather than coupled two-electron transfers. Mutational analysis of active site amino acids reveals their role in governing proton coupled redox events, which likely involves a hydrogen bonding network consisting of these residues and water molecules. Additionally, steady state kinetics assays coupled to site-directed mutagenesis of M. tuberculosis sulfite reductase identify a tyrosine residue adjacent to the active site which partially controls substrate preference, by influencing the electronic environment of the active site siroheme cofactor. Stopped-flow absorbance spectroscopy and rapid freeze quench electron paramagnetic resonance studies provide a first glimpse of a potential reaction intermediate during reduction of sulfite by sulfite reductase. Overall, our fundamental understanding of how sulfite and nitrite reductase enzymes catalyze complex multi-electron multi-proton reactions is advanced, and insight into the different approaches Nature employs to govern such powerful chemistry is revealed.

Heme containing proteins and their reactivity play a central role in biological systems; they have a vast range of functions including electron transfer, catalysis, and respiration. Cytochromes c and heme c containing proteins have been used widely as model systems to understand how structure and dynamics lead toward function. In this thesis, a variety of biophysical methods are used to investigate two heme c containing model systems to gain insight into how redox potential and reactivity are modulated through changes in the local environment. Mitochondrial cytochrome c undergoes several pH dependent conformational rearrangements that involve different heme ligation and have associated changes in redox potential. Under basic conditions (pH greater than 8), the axial methionine (Met) residue is replaced by one of several nitrogen based ligands, usually a nearby lysine residue, and is coined the "alkaline transition". It is accompanied by a large downward shift in redox potential. The functional utility of this conformational change is not fully understood however it is strongly implicated in the signaling cascade for apoptosis. Bacterial monoheme cytochromes c exhibit similar phenomenological Met-loss behavior as a function of electrode material. In Chapter 2 we utilize Hydrogenobacter thermophilus cytochrome c552 as a model system for the assessment of redox thermodynamics and changes in redox potential associated with the Met-loss form. In Chapter 3 we extend our investigation to homologous cytochromes c. Bacterial cytochrome c peroxidases catalyze the two-electron reduction of hydrogen peroxide to water utilizing cytochrome c as an endogenous electron donor. Chapter 4 describes the first recombinant construct of the diheme Nitrosomonas europaea cytochrome c peroxidase (Ne CCP); a defining family member of constitutively active cytochrome c peroxidases. A variety of biophysical techniques were used to confirm similarity between the recombinant Ne CCP and native enzyme. Chapter 5 extends our investigation to the role of constitutively conserved glutamine and glutamic acid residues within the active site, and two conserved tryptophan residues; the first situated between hemes and the second distal to the active site. In Chapter 6, stopped flow spectroscopy is used to investigate the first intermediates of the Ne CCP catalytic mechanism.