Asha Rajapakshe

Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase

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, C., et al. (1997) Cell 91, 973-983], which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 A and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into greater proximity have been proposed [Feng, C., et al. (2003) Biochemistry 42, 5816-5821] to explain the relatively rapid IET kinetics, which are much faster than those theoretically predicted from the crystal structure. To explore the proposed role(s) of the tether in facilitating this conformational change, we altered both its length and flexibility 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 discernible systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a 3-fold decrease in the IET rate constant. Deletions of nonconserved 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 steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a 5-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.

Determination of the Distance between the Mo(V) and Fe(III) Heme Centers of Wild Type Human Sulfite Oxidase by Pulsed EPR Spectroscopy

Intramolecular electron transfer (IET) between the molybdenum and heme centers of vertebrate sulfite oxidase (SO) is proposed to be a key step in the catalytic cycle of the enzyme. However, the X-ray crystallographic distance between these centers, R(MoFe) = 32.3 Å, appears to be too long for the rapid IET rates observed in liquid solution. The Mo and heme domains are linked by a flexible tether, and it has been proposed that dynamic interdomain motion brings the two metal centers closer together and thereby facilitates rapid IET. To date, there have been no direct distance measurements for SO in solution that would support or contradict this model. In this work, pulsed electron-electron double resonance (ELDOR) and relaxation induced dipolar modulation enhancement (RIDME) techniques were used to obtain information about R(MoFe) in the Mo(V)Fe(III) state of wild type recombinant human SO in frozen glassy solution. Surprisingly, the data obtained suggest a fixed structure with R(MoFe) = 32 Å, similar to that determined by X-ray crystallography for chicken SO, although the orientation of the R(MoFe) radius-vector with respect to the heme center was found to be somewhat different. The implications of these findings for the flexible tether model are discussed.

Characterization of Chloride-Depleted Human Sulfite Oxidase by Electron Paramagnetic Resonance Spectroscopy: Experimental Evidence for the Role of Anions in Product Release

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 >9 exhibits characteristic EPR spectra that correspond to two structurally different forms of the Mo(V) active center termed 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 on the presence of chloride. In this work, we have prepared and purified samples of the wild type and various mutants of human SO that are depleted of chloride. These samples do not exhibit the typical lpH EPR spectrum at low pH but rather exhibit 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.

Intramolecular electron transfer in sulfite-oxidizing enzymes: probing the role of aromatic amino acids

Sulfite oxidase (SO) is a molybdoheme enzyme that is important in sulfur catabolism, and mutations in the active site region are known to cause SO deficiency disorder in humans. This investigation probes the effects that mutating aromatic residues (Y273, W338, and H337) in the molybdenum-containing domain of human SO have on both the intramolecular electron transfer (IET) rate between the molybdenum and iron centers using laser flash photolysis and on catalytic turnover via steady-state kinetic analysis. The W338 and H337 mutants show large decreases in their IET rate constants (kET) relative to the wild-type values, suggesting the importance of these residues for rapid IET. In contrast, these mutants are catalytically competent and exhibit higher kcat values than their corresponding kET, implying that these two processes involve different conformational states of the protein. Redox potential investigations using spectroelectrochemistry revealed that these aromatic residues close to the molybdenum center affect the potential of the presumably distant heme center in the resting state (as shown by the crystal structure of chicken SO), suggesting that the heme may be interacting with these residues during IET and/or catalytic turnover. These combined results suggest that in solution human SO may adopt different conformations for IET and for catalysis in the presence of the substrate. For IET the H337/W338 surface residues may serve as an alternative-docking site for the heme domain. The similarities between the mutant and wild-type EPR spectra indicate that the active site geometry around the Mo(V) center is not changed by the mutations studied here.

Structural studies of the molybdenum center of mitochondrial amidoxime reducing component (mARC) by pulsed EPR spectroscopy and 17O-labeling.

Mitochondrial amidoxime reducing components (mARC-1 and mARC-2) represent a novel group of Mo-containing enzymes in eukaryotes. These proteins form the catalytic part of a three-component enzyme complex known to be responsible for the reductive activation of several N-hydroxylated prodrugs. No X-ray crystal structures are available for these enzymes as yet. A previous biochemical investigation [Wahl, B., et al. (2010) J. Biol. Chem., 285, 37847-37859 ] has revealed that two of the Mo coordination positions are occupied by sulfur atoms from a pyranopterindithiolate (molybdopterin, MPT) cofactor. In this work, we have used continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy and density functional theoretical (DFT) calculations to determine the nature of remaining ligands in the Mo(V) state of the active site of mARC-2. Experiments with samples in D(2)O have identified the exchangeable equatorial ligand as a hydroxyl group. Experiments on samples in H(2)(17)O-enriched buffer have shown the presence of a slowly exchangeable axial oxo ligand. Comparison of the experimental (1)H and (17)O hyperfine interactions with those calculated using DFT has shown that the remaining nonexchangeable equatorial ligand is, most likely, protein-derived and that the possibility of an equatorial oxo ligand can be excluded.

Kinetic and thermodynamic effects of mutations of human sulfite oxidase.

Animal SOs possess two redox-active domains and are ideal for studying IET in relation to the overall conformation of the protein and the molecular dynamics of the motion of the two domains relative to one another. The procedures for preparing and purifying recombinant hSO [34, 44] have been further optimized in our laboratory [4, 5, 29], so that a wide range of mutant proteins in 50–100 mg quantities can be prepared for comprehensive kinetic, mechanistic, and spectroscopic studies. The ready accessibility of recombinant hSO has made it possible to investigate the effects of modifying amino acid residues of interest to better understand the long range IET process in hSO and to obtain insight into biochemical basis of pathological mutations of hSO. Experimental and theoretical studies agree that domain movement is necessary for SO to achieve a productive conformation during electron transfer, and that the length and composition of the interdomain tether is an important factor in the rates of IET. However, the interactions such as hydrogen bonding and hydrophobic effects involved in mediating the correct mutual orientation and the distance following the initial orientation are unknown. Studies of site-specific mutants reveal a great deal about the IET complex in hSO. Mutating several aromatic residues that extend from the pterin to the surface of the protein suggest that alternative ET pathways may be in operation in hSO. The profound effects of mutating residues surrounding the Mo active site (e. g. R160Q and Y343F) have been demonstrated [28, 45], and extension to mutations of other positively charged residues surrounding the active site, such as Lys200, is warranted. Finally, all of the discussions presented here and elsewhere for IET in hSO rest upon the single structure of intact native cSO. It is clear that X-ray crystal structures of hSO and various mutants are needed to better understand the unexpected properties of this fascinating biomedically important protein.