Equilibria amongst different molybdenum (V)-containing species from sulphite oxidase. Evidence for a halide ligand of molybdenum in the low-pH species

Abstract

The interaction of chloride, fluoride and phosphate ions with the molybdenum centre of sulphite oxidase in the pH range 6.2 to 9.6 has been studied by e.p.r. of Mo(V) in the enzyme reduced by sulphite. Detailed studies were made from e.p.r. spectra recorded at about 120K and more limited studies from spectra of liquid samples at about 295K and also from enzyme activity measurements. Interconversion between low-pH and high-pH Mo(V) e.p.r. signal-giving species [described by Lamy, Gutteridge & Bray (1980) Biochem. J. 185, 397-403] is influenced by chloride concentration, a 10-fold increase in concentration (in the range of about 1 mM to 100 mM) causing an increase of about 1 pH unit in the apparent pK for the conversion. This suggests that chloride is a constituent of the low-pH species. In support of this, high concentrations of fluoride modified the e.p.r. spectrum. Partial conversion to a Mo(V) species, in which F- has presumably replaced Cl- and showing hyperfine coupling of A(19F)av. 0.5mT, is indicated. It is proposed that interconversion between high-pH and low-pH species is of the form: (formula; see text) No evidence that Cl- is essential for enzymic activity was found. Data relating to equilibria amongst low-pH, high-pH and also the phosphate species are presented. Depending on pH and on concentrations of Cl- and H2PO4-, one, two, or all three species may be present. Qualitatively, under appropriate conditions, the phosphate species tends to replace some or all of the low-pH species. Quantitative analysis by a computer procedure permitted an appropriate scheme to be deduced and equilibrium constants to be evaluated. Studies on the e.p.r. signals at 295K indicated that similar equilibria applied in liquid solution, but with some changes in the values of the constants. The structure of the molybdenum centre in its various states and the nature of the enzymic reaction are discussed.

MoV Electron Paramagnetic Resonance of Sulfite Oxidase Revisited: The Low-pH Chloride Signal

“…Previous work has shown that the addition of chloride promotes the conversion of the high-pH species to the low-pH species. 13 Moreover, Bray and co-workers found that increasing the chloride concentration by a factor of 10 shifted the pK a of the interconversion between high-and low-pH signal-giving species by one pH unit. 13 In addition, they reported that the low-pH EPR signals were identical in the absence and presence of chloride and attributed this to trace amounts of contaminating chloride in their "chloride-free" buffers.…”

“…13 Moreover, Bray and co-workers found that increasing the chloride concentration by a factor of 10 shifted the pK a of the interconversion between high-and low-pH signal-giving species by one pH unit. 13 In addition, they reported that the low-pH EPR signals were identical in the absence and presence of chloride and attributed this to trace amounts of contaminating chloride in their "chloride-free" buffers. 25 Figure 1b and c compare the low-pH spectra obtained in the presence and in the rigorous absence of added chloride.…”

“…Recently, efforts have been focused on studying SO using advanced pulsed EPR techniques, such as electron-nuclear double resonance (ENDOR) and electron-spin envelope echo modulation (ESEEM), 9 but the conventional continuous-wave (CW) EPR has not been re-evaluated in nearly two decades. In this work, 10 we revisit the Mo V EPR of one of the major signals from this important enzyme, [11][12][13] and using the stable magnetic isotopes 35 Cl and 37 Cl, in conjunction with difference EPR spectroscopy, and analogous complexes with other halides, we present new evidence for halide molybdenum coordination of the Mo V low-pH species.…”

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

“…a SAD, b NITRATE, c PERCHLORATE, and d CYANIDE, The2mFo-DFc electron density maps contoured at 1r (blue) and the mFo-DFc electron density maps contoured at 3r (red) calculated in the absence of a sixth ligand (left) and calculated in the presence of the sulfur atom in the final refinement (right) g n * 0.5 for 35,37 Cl and I = 1/2, g n = 2.26 for 31 P) and hence superhyperfine structure from these nuclei might be observed in the EPR spectra associated with Mo(V) species. Hyperfine couplings associated with chloride nuclei in equatorial positions have been reported in model complexes of Mo(V) ions in nearly octahedral or squarepyramidal coordination [24] and in the molybdenum enzyme sulfite oxidase [25]. Larger couplings should be expected for the phosphorous atom because of the higher nuclear g factor (g n = 2.26).…”

Modified Active Site Coordination in a Clinical Mutant of Sulfite Oxidase

“…The high-pH signal occurs at low anion concentrations and at high-pH values, while the low-pH/Cl -signal occurs at low pH and high chloride. 37,38 A third category of signals are those from oxy-anion complexes, which tend to form at low pH and high oxy-anion concentrations. SO Mo V EPR signals arising from anion accessed by following the link in the citation at the bottom of the page.…”