Molecular Basis for Enzymatic Sulfite Oxidation

Abstract

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased K m(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. R55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDH R55M . Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.Sulfite-oxidizing enzymes protect cells against potentially fatal damage to DNA and proteins caused by exposure to sulfite, and consequently they are found in all forms of life (1). In bacteria, sulfite oxidation is often linked to energy-generating processes during chemolithotrophic growth on reduced sulfur compounds (2, 3), whereas both plant and vertebrate sulfite oxidases have been shown to detoxify sulfite arising from the degradation of methionine and cysteine and exposure to sulfur dioxide (4, 5).All known sulfite-oxidizing enzymes belong to the same family of mononuclear molybdenum enzymes. Their active sites contain one molybdopterin unit per molybdenum atom, and these enzymes may also contain heme groups as accessory redox centers (6 -9). Examples of different types of sulfite-oxidizing molybdoenzymes are the homodimeric plant sulfite oxidase, which does not contain a heme group and uses oxygen as its preferred electron acceptor (9), the homodimeric chicken and human liver sulfite oxidases (CSO 3 and HSO, respectively) (10), which are also able to use oxygen as an electron acceptor, and the bacterial sulfite dehydrogenase (SDH) isolated from the soil bacterium Starkeya novella (11, 12), which cannot donate electrons directly to oxygen. Each monomer of CSO and HSO contains a heme b center in addition to the molybdenum center, and the redox centers are located within separate, flexibly linked domains of the same protein subunit. In contrast, the bacterial enzyme is a heterodimer where each subunit of the enzyme contains one redox center. The molybdopterin cofactor is located in the larger 40.2-kDa SorA subunit, and the c-type heme is located in the smaller, 8.8-kDa SorB subunit (12). The SDH quaternary structure thus differs clearly from that of the human and chicken sulfite oxidases.Crystal structures are available for plant sulfite oxidase, CSO, and the bacterial SDH (10,11,(13)(14)(15) and have revealed molecular details of the sulfite-oxidizing enzymes. In the CSO structure, the mobile heme b domain occupies a position too removed from the...

The Mononuclear Molybdenum Enzymes

“…An R55Q mutant of the bacterial enzyme is not compromised in intramolecular electron transfer, however, 325 suggesting that the role of the arginine in the human enzyme has more to do with properly orienting the heme domain for electron transfer rather than directly participating in a specific electron transfer pathway. The R55Q mutant of the bacterial enzyme is significantly compromised in substrate binding 285 and hydrolysis of the Mo IV ·sulfate complex, resulting in accumulation of the blocked EPR species discussed above in regard to the eukaryotic enzymes; 326 in the crystal structure of the R55 M mutant, 285 sulfate is coordinated to the molybdenum at the equatorial position otherwise occupied by the catalytically labile Mo=O, directly supporting the proposed structure for the blocked enzyme. Thus, as with the vertebrate enzymes, this arginine appears to be involved in facilitating hydrolysis of the E red ·SO 4 2− complex and product dissociation from the molybdenum center.…”

“…It is worth noting that the corresponding Arg 55 in the closely related sulfite dehydrogenase from Starkeya novella has also been mutated to Met with results comparable to those seen with the chicken enzyme: the effect on the limiting rate constant for enzyme reduction at high [sulfite] is modest, but K d increases 3 orders of magnitude. 285 …”

Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella

“…In the fully oxidized Mo(VI) state this equatorial ligand is an oxo group [9]. In the generally accepted catalytic mechanism [10, 11], this oxo ligand is attacked by sulfite to form a sulfate-bound Mo(IV) intermediate [10]. Subsequent hydrolysis of this intermediate and one-electron oxidation lead to Mo(V)–OH species that can be studied by electron paramagnetic resonance (EPR) spectroscopy [12, 13].…”

“…Two mutations are presented, SDH R55Q (analogous to HSO R160Q ), where the hydrophilic positively charged arginine was changed to a neutral hydrophilic glutamine, and SDH R55M , where the change was to the neutral, but hydrophobic, methionine. Comprehensive studies of the overall reaction kinetics and intramolecular electron transfer kinetics for SDH R55M and SDH R55Q have also recently been reported [11, 12]. …”

Metabolic adaptation and trophic strategies of soil bacteria—C1- metabolism and sulfur chemolithotrophy in Starkeya novella

“…A Sox complex independent sulfite oxidizing enzyme, SorAB, had already been identified previously (Kappler et al, 2000) and the enzyme itself has been extensively characterized (Kappler and Bailey, 2005; Kappler et al, 2006, 2012; Rapson et al, 2008; Bailey et al, 2009; Emesh et al, 2009). No evidence was found for the presence of Sox complex independent sulfide oxidizing enzymes such as flavocytochrome c and sulfide:quinone reductase.…”