The Mo(V) state of the molybdoenzyme sulfite oxidase (SO) is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Vertebrate SO at pH < 7 and pH > 9 exhibits characteristic EPR spectra that correspond to two structurally different forms of the Mo(V) active center referred to as the low-pH (lpH) and high-pH (hpH) forms, respectively. Both EPR forms have an exchangeable equatorial OH ligand, but its orientation in the two forms is different. It has been hypothesized that the formation of the lpH species is dependent upon the presence of chloride. In this work we have prepared and purified samples of wild type and various mutants of human SO that are depleted in chloride. These samples do not exhibit the typical lpH EPR spectrum at low pH, but rather show spectra that are characteristic of the blocked species that contains an exchangeable equatorial sulfate ligand. Addition of chloride to these samples results in the disappearance of the blocked species and the formation of the lpH species. Similarly, if chloride is added before sulfite, the lpH species is formed instead of the blocked one. Qualitatively similar results were observed for samples of sulfite oxidizing enzymes from other organisms that were previously reported to form a blocked species at low pH. However, the depletion of chloride has no effect upon the formation of the hpH species.The sulfite-oxidizing enzymes (SOEs), represented by sulfite oxidase (SO) in vertebrates and plants and sulfite dehydrogenase (SDH) in bacteria, catalyze the oxidation of sulfite to sulfate as represented by generic Eq. 1 (1).(1)In humans SO is essential for normal neonatal neurological development, and inborn deficiencies in SO result in severe physical and neurological disorders and early death (2,3).Reaction (1) is catalyzed by the square-pyramidal oxo-molybdenum active center, which has three equatorial sulfur ligands (one from the conserved cysteinyl side chain, and two from the molybdopterin cofactor), one axial oxo ligand, and an exchangeable equatorial oxo ligand in the solvent accessible pocket of the active site (4, 5). During the proposed catalytic cycle (6), sulfite initially reduces Mo(VI) to Mo(IV). Regeneration of the Mo(VI) state Unlike X-ray crystallography or extended X-ray absorption fine structure (EXAFS) spectroscopy, EPR can detect protons in the vicinity of a paramagnetic center and is able to unequivocally identify specific nuclei through using substitutions by or permutations of magnetic isotopes (e.g., 16 O → 17 O, 35 Cl → 37 Cl, 14 N → 15 N, etc.). Both, continuous wave (CW) and pulsed EPR spectroscopic approaches have been used to establish the effects of pH, anions in the media, and mutations near the active site on the identity and structure of the exchangeable equatorial ligand of the Mo(V) ion. It was found that in the absence of inhibiting anions (e.g., PO 4 3− , AsO 4 3− ), wild type (wt) vertebrate SO can show two distinct types of EPR signals, high-pH (hpH) and low-pH (lpH), corresponding to two ...

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO (Kisker et al., Cell, 1997, 91, 973-983), which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 Å, and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into closer proximity have been proposed (Feng et al., Biochemistry, 2003, 41, 5816-21) to explain the relatively rapid IET kinetics, which are much faster than theoretically predicted from the crystal structure. In order to explore the proposed role(s) of the tether in facilitating this conformational change, both its length and flexibility were altered in HSO by site-specific mutagenesis and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernable systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a three-fold decrease in the IET rate constant. Deletions of non-conserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steadystate kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a five-fold increase in the sulfite K m value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these Proline to Alanine and deletion mutants are identical to those of wild type HSO, indicating no significant change in the Mo active site geometry.Sulfite oxidase (SO) catalyzes the oxidation of sulfite to sulfate, using oxidized ferricytochrome c (cyt c ox ) as the physiological electron acceptor (eq. 1) (1-4). This reaction is biologically essential, serving as the final step in the catabolism of sulfur containing amino acids, methionine and cysteine, and as a detoxification mechanism for sulfite. † This research was supported by NIH Grant GM-037773 (to JHE); Ruth L. Kirchstein-NIH Fellowship 1F32GM082136-01 (to KJW) *To whom correspondence should be addressed. J.H.E.: jenemark@u.arizona.edu; phone, (520) 621-2245; fax, (520) 626-8065. G.T.:, gtollin@u.arizona.edu; phone, (520) 621-3447; fax, (520) 621-9288. Supporting Information Available: Primer design; iron to molybdenum ratios determined using inductively coupled plasma; and laser flash photolysis results for proline to alanine ...

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Sulfite oxidase (SO) is a molybdenum-cofactor-dependent enzyme that catalyzes the oxidation of sulfite to sulfate, the final step in the catabolism of the sulfur-containing amino acids, cysteine and methionine. The catalytic mechanism of vertebrate SO involves intramolecular electron transfer (IET) from molybdenum to the integral b-type heme of SO and then to exogenous cytochrome c. However, the crystal structure of chicken sulfite oxidase (CSO) has shown that there is a 32 Å distance between the Fe and Mo atoms of the respective heme and molybdenum domains, which are connected by a flexible polypeptide tether. This distance is too long to be consistent with the measured IET rates. Previous studies have shown that IET is viscosity dependent (Feng et al., Biochemistry, 2002, 41, 5816) and also dependent upon the flexibility and length of the tether (Johnson-Winters et al., Biochemistry, 2010, 49, 1290). Since IET in CSO is more rapid than in human sulfite oxidase (HSO) (Feng et al., Biochemistry, 2003, 42, 12235) the tether sequence of HSO has been mutated into that of CSO, and the resultant chimeric HSO enzyme investigated by laser flash photolysis and steady-state kinetics in order to study the specificity of the tether sequence of SO on the kinetic properties. Surprisingly, the IET kinetics of the chimeric HSO protein with the CSO tether sequence are slower than wildtype HSO. This observation raises the possibility that the composition of the non-conserved tether sequence of animal SOs may be optimized for individual species.