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 ...

All reported sulfite oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. In order to better understand the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions, R55Q and R55M, as well as the positively charged substitution, R55K, in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for increasing the understanding of residue function in IET without the added complexity of the inter-domain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) showed increased IET rate constants relative to the wildtype (3–4 fold) when studied by laser flash photolysis. The gain in function observed in SDHR55Q and SDHR55M suggests that the reduction of IET seen in HSOR160Q is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the inter-domain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDHwt, SDHR55M, and SDHR55Q show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (Keq) and rate constants (ket) of both variants with respect to SDHWT enzyme. SDHWT has a ket that is independent of pH and a Keq that increases as pH decreases, whereas both SDHR55M and SDHR55Q have a ket that increases as pH decreases, and SDHR55M has a Keq that is pH independent. IET in the SDHR55Q variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from SDHWT, but which also occurs in HSO. IET in SDHR55K is slower than for SDHWT. A new analysis of the possible mechanistic pathways for sulfite oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.