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.

Methanogens are a group of microorganisms that utilize simple compounds such as H₂ + CO₂, acetate and methanol for the production of methane, an end-product of their metabolism.  These obligate anaerobes belonging to the archaeal domain inhabit diverse anoxic environments such as rice paddy fields, human guts, rumen of ruminants, and hydrothermal vents.  In these habitats, methanogens are often exposed to O₂ and previous studies have shown that many methanogens are able to tolerate O2 exposure.  Hence, methanogens must have developed survival strategies to be able to live under oxidative stress conditions.  The anaerobic species that lived on Earth during the early oxygenation event were first to face oxidative stress.  Presumably some of the strategies employed by extant methanogens for combating oxidative stress were developed on early Earth.   Our laboratory is interested in studying the mechanism underlying the oxygen tolerance and oxidative stress responses in methanogenic archaea, which are obligate anaerobe.  Our research concerns two aspects of oxidative stress.  (i) Responses toward extracellular toxic species such as SO32-, that forms as a result of reactions of O₂ with reduced compounds in the environment.  These species are mostly seen in anaerobic environments upon O₂ exposure due to the abundance of reduced components therein.  (ii) Responses toward intracellular toxic species such as superoxide and hydrogen peroxide that are generated upon entry of O₂ and subsequent reaction of O₂ with reduced component inside the cell.  Aerobic microorganisms experience the second problem.  Since a large number of microorganisms of Earth are anaerobes and the oxidative defense mechanisms of anaerobes are relatively less studied, the research in our laboratory has focused on this area.  My thesis research covers two studies that fall in the above-mentioned two focus areas. In 2005-2007 our laboratory discovered that certain methanogens use an unusual sulfite reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO32- that is produced outside the cell from a reaction between oxygen and sulfide.  This reaction occurred during early oxygenation of Earth and continues to occur in deep-sea hydrothermal vents.  Fsr, a flavoprotein, carries out a 6-electron reduction of SO32- to S2-.  It is a chimeric protein where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively.  We hypothesized that Fsr was developed in a methanogen from pre-existing parts.  To begin testing this hypothesis we have carried out bioinformatics analyses of methanogen genomes and found that both Fsr-N homologs and Fsr-C homologs are abundant in methanogens.  We called the Fsr-C homolog dissimilatory sulfite reductase-like protein (Dsr-LP).  Thus, Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP) in methanogens.  During the course of this study, we also identified two new putative F420H2-dependent enzymes, namely F420H2-dependent glutamate synthase and assimilatory sulfite reductase. Another aspect of my research concerns the reactivation of proteins that are deactivated by the entry of oxygen inside the cell.  Here I focused specifically on the role of thioredoxin (Trx) in methanogens.  Trx, a small redox regulatory protein, is ubiquitous in all living cells.  In bacteria and eukarya, Trx regulates a wide variety of cellular processes including cell divison, biosynthesis and oxidative stress response.  Though some Trxs of methanogens have been structurally and biochemically characterized, their physiological roles in these organisms are unknown.  Our bioinformatics analysis suggested that Trx is ubiquitous in methanogens and the pattern of its distribution in various phylogenetic classes paralleled the respective evolutionary histories and metabolic versatilities.  Using a proteomics approach, we have identified 155 Trx targets in a hyperthermophilic phylogenetically deeply-rooted methanogen, Methanocaldococcus jannaschii.  Our analysis of two of these targets employing biochemical assays suggested that Trx is needed for reactivation of oxidatively deactivated enzymes in M. jannaschii.  To our knowledge, this is the first report on the role of Trx in an organism from the archaeal domain. During the course of our work on methanogen Trxs, we investigated the evolutionary histories of different Trx systems that are composed of Trxs and cognate Trx reductases.  In collaboration with other laboratories, we conducted bioinformatics analysis for the distribution of one of such systems, ferredoxin-dependent thioredoxin reductase (FTR), in all organisms.  We found that FTR was most likely originated in the phylogenetically deeply-rooted microaerophilic bacteria where it regulates CO₂ fixation via the reverse citric acid cycle. / Ph. D.