Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella
Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDHR55M) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053\xe2\x80\x932063, 2009). For SDHR55M only the high-pH form is observed, and samples prepared in H217O-enriched buffer show essentially the same 17O hyperfine interaction and nuclear quadrupole interaction parameters as SDHWT enzyme. However, the pH dependence of the EPR spectra of SDHR55Q, in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDHWT. For SDHR55Q the blocked form with bound sulfate is generated at low pH, as verified by 33S couplings observed upon reduction with 33S-labeled sulfite. This observation of bound sulfate for SDHR55Q supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156\xe2\x80\x932163, 2009). At pH \xe2\x89\xa5 8 the high-pH form dominates for SDHR55Q.
Introduction<!>Materials and methods<!>Continuous-wave electron paramagnetic resonance<!>Exchangeable equatorial ligands in SDHR55M and high-pH SDHR55Q<!>Exchangeable ligand for SDHR55Q at low pH<!>17O electron spin echo envelope modulation<!>Neighboring exchangeable proton in SDHR55Q at low pH<!>Discussion<!>Conclusion<!>
<p>Sulfite-oxidizing enzymes (SOEs) occur in vertebrates, plants, and certain bacteria [1]. In vertebrate sulfur metabolism, sulfite oxidase (SO) converts toxic sulfite to sulfate in the final degradation step of the sulfur-containing amino acids cysteine and methionine [2, 3]. Sulfite dehydrogenase (SDH), isolated from the soil bacterium Starkeya novella, oxidizes sulfite during chemolithotrophic growth with thiosulfate as an energy source [4, 5]. Despite their different metabolic roles, both SO and SDH have nearly identical active-site geometries [6–8]. Their square-pyramidal oxomolybdenum active sites have an axial oxo ligand, one equatorial sulfur from a cysteine amino acid residue, two equatorial sulfurs from the molybdopterin cofactor, and one exchangeable equatorial ligand that undergoes changes that depend on the stage of the catalytic cycle. 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].</p><p>Numerous studies of the paramagnetic Mo(V) centers of SOEs from various sources by continuous-wave (CW) and pulsed EPR techniques have revealed three distinct types of EPR spectra corresponding to different structural forms of the active center: high-pH; low-pH; and blocked [13]. For the high-pH and low-pH forms the exchangeable equatorial ligand is OH [14–19].</p><p>The spectral features of the high-pH, low-pH, and blocked forms are remarkably independent of the organism; however, the particular pH dependence of interconversions among these forms can depend upon the organism, specific mutations near the active site, and the mode of reduction. For example, for plant SO from Arabidopsis thaliana (At-SO) reduction by sulfite at low pH produces the blocked form in which sulfate (instead of OH) is coordinated to Mo(V), whereas reduction by titanium(III) citrate generates the low-pH form [19]. The EPR spectrum of the Mo(V) center in SDHWT is independent of pH within the studied range from 4.9 to 10, and only a high-pH-type spectrum is observed [20, 21]. However, substitution of the active-site tyrosine by phenylalanine (SDHY236F) results in generation of the low-pH form at pH ≤ 7 [22]. Thus, this substitution results in an effective lowering of the local pH. A qualitatively similar lowering of the local pH was obtained for the analogous mutant in human SO (HSOY343F), where the formation of the low-pH species has shifted to pH ~ 8, a typical value for observation of the high-pH form [23]. For HSOY343F only insignificant amounts of the high-pH form were generated, even at pH 10. At pH < 7 the blocked form was observed for HSOY343F [23]. For vertebrate SOs, the presence of certain anions (e.g., PO43−, AsO43−) produces additional EPR spectral forms [24–26].</p><p>Recently, we have investigated the pathogenic mutant, HSOR160Q, in which an active-site arginine that is conserved in all SOEs is replaced by glutamine [27]. The high-pH form was observed at high pH (above 8). However, at pH < 7 a blocked form was generated that had a spectrum different from the spectra observed earlier in plant SO (At-SO) and HSOY343F. The electron spin echo envelope modulation (ESEEM) investigation of the blocked form of HSOR160Q yielded nuclear quadrupole interaction (NQI) parameters of the 17O oxo ligand that were interpreted as indicating a six-coordinate Mo center with weak axial coordination trans to the oxo ligand by the substituted glutamine residue [27, 28]. Additionally, pulsed electron–nuclear double resonance (ENDOR) experiments revealed the presence of a nearby (R ~ 3.1 Å) weakly coupled exchangeable proton that was suggested to be hydrogen-bonded to the oxygen of the equatorial sulfate ligand. At intermediate pH values (6.5–8) a completely new Mo(V) signal was observed (denoted as species 2 in [27]). The structure of species 2 is not yet known, but it is also the major EPR-active product generated upon reduction of HSOR160Q by titanium(III) citrate [27]. This result for HSOR160Q contrasts with the reduction of At-SO by titanium(III) citrate, which gives a typical low-pH signal [19].</p><p>It was not clear whether the unusual spectroscopic properties of HSOR160Q were organism-specific or mutation-specific. Therefore, in this work we have investigated mutations of the analogous active-site arginine (R55) for an enzyme from a completely different organism, namely, the bacterial SDH from Starkeya novella. Two mutations are presented, SDHR55Q (analogous to HSOR160Q), where the hydrophilic positively charged arginine was changed to a neutral hydrophilic glutamine, and SDHR55M, where the change was to the neutral, but hydrophobic, methionine. Comprehensive studies of the overall reaction kinetics and intramolecular electron transfer kinetics for SDHR55M and SDHR55Q have also recently been reported [11, 12].</p><!><p>SDHR55M and SDHR55Q were generated as previously described [11, 12], expressed in Rhodobacter capsulatus, and purified using previously developed procedures for SDH proteins [29].</p><p>The low pH (pH 5.8–6.0) samples of SDHR55M and SDHR55Q were prepared using 50 mM bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis–Tris) buffer. The enzyme samples (474 μM of SDHR55M and 481 μM of SDHR55Q) were reduced with a 20-fold excess of sodium sulfite under argon and immediately frozen in liquid nitrogen. The same buffer system and procedure were used for reduction with 33S-labeled sulfite, prepared as previously described [30].</p><p>The low pH samples in H217O-enriched water were prepared by first concentrating a 100 μL solution of the enzyme in 100 mM Bis–Tris at pH 5.8 to reduce the amount of H216O. Next, a solution of 100 mM Bis–Tris buffer (pH 5.8) was vacuum-centrifuged to evaporate the H216O, and the pelleted buffer was redissolved in the same volume of H217O. The concentrated enzyme sample was then incubated in 30 μL of the buffer prepared with H217O for approximately 3 h at 277 K. Finally, the enzyme was reduced with a 20-fold excess of sodium sulfite under argon and then immediately frozen in liquid nitrogen.</p><p>For titanium(III) citrate reduced low-pH samples of SDH in H2O or D2O, a stock solution of titanium(III) citrate was diluted to 3.76 mM in 100 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis–Tris propane) (pH 6.0). Approximately 850 μM SDHR55Q (22 μL) in 50 mM Bis–Tris propane was injected into an EPR tube, which was then placed inside a Schlenk tube. The Schlenk tube was pumped to obtain a mild vacuum and then purged with argon. This procedure was repeated several times over a period of 40 min to degas the protein. Next, 5 μL of the diluted titanium(III) citrate solution (700 μM final concentration) in well-degassed 100 mM Bis–Tris propane buffer (pH 6.0) was injected into the protein solution under argon, and the reduced SDHR55Q enzyme was frozen in liquid nitrogen immediately. The final concentration ratio of SDHR55Q to titanium(III) citrate was approximately 2:1.</p><p>The X-band CW EPR experiments were performed using a Bruker ESP-300 EPR spectrometer at 77 K. The resonator used in these experiments was a standard rectangular cavity operated in TE102 mode. The principal g values of the Mo(V) center were determined using a 1,1-diphenyl-2-picrylhydrazyl free radical standard (g = 2.0036).</p><p>The pulsed EPR experiments were performed using home-built broadband pulsed EPR spectrometers operating at the microwave frequencies from 8 to 18 GHz (X and Ku microwave bands) (http://quiz2.chem.arizona.edu/epr) and from 26 to 40 GHz (Ka band) [31]. The measurement temperature was about 21 K. The pulse sequences used for various ESEEM and pulsed ENDOR experiments are described in the electronic supplementary material.</p><!><p>Previous studies have shown that the CW EPR spectrum of SDHWT is independent of pH [21], unlike the spectra of plant and animal SOEs, and the SDHWT spectrum is characteristic of the high-pH form of eukaryotic SOEs [13]. SDHR55M gives EPR spectra similar to those of SDHWT, and traces 1 and 2 in Fig. 1 show the spectra recorded at pH 9.0 and 5.8, respectively. The principal g values determined from these spectra are typical for the high-pH form of SOEs (Table 1).</p><p>In contrast, the EPR spectra of SDHR55Q are distinctly different at low and high pH. The spectrum observed at high pH (trace 3 in Fig. 1, pH 9.0) is similar to the usual high-pH spectrum of all SOEs. However, the spectrum observed at low pH (trace 4 in Fig. 1, pH 5.8) represents a mixture of two species. The major species (approximately 75%, as estimated by subtracting the appropriately normalized high-pH spectrum of trace 3 and double-integrating the EPR spectra), species 1, is similar to the blocked form (large gz and no observable hyperfine splittings), whereas the minor species (approximately 25%) is similar to the high-pH form. Decreasing the pH from 5.8 to 4.9 did not change the relative amounts of these two forms. However, in the intermediate-pH range (6–9) the amount of species 1 decreased, and the amount of the high-pH-like species increased with increasing pH.</p><p>Apart from the major EPR lines (marked by asterisks and diamonds in Fig. 1) corresponding to the Mo(V) centers containing nonmagnetic molybdenum isotopes, there are several minor features seen in the EPR spectra of Fig. 1 (e.g., at 336.5 and 342.5 mT in traces 1–3, and at 349 mT in all traces) corresponding to Mo(V) centers containing magnetic molybdenum isotopes (95Mo and 97Mo, both I = 5/2, total natural abundance approximately 25.5%). Out of these, the features at 336.5 and 342.5 mT in the spectra of SDHR55M (traces 1 and 2) are at similar positions to the gz and gy turning points of species 1 of SDHR55Q seen in trace 4. Assuming that a similar species also contributes to the spectra of SDHR55M, we estimated its yield by double integration of spectra 1 and 2, which resulted in a negligible amount of approximately 5%. However, even such a minor contribution is unlikely for SDHR55M because the mentioned spectral features are observed both at low pH and at high pH, whereas all of the known blocked species (to which species 1 actually belongs, as shown by the pulsed EPR experiments described later) only form at low pH. We conclude, therefore, that the spectra of SDHR55M correspond to a single high-pH-like species. Additional evidence in support of this conclusion comes from the EPR spectra of SDHR55M in D2O buffer (see the electronic supplementary information).</p><p>Reduction of SDHR55Q with titanium(III) citrate instead of sulfite at pH 5.8 produced only a very weak EPR signal of the high-pH-like form. This result contrasts with Ti(III) reductions of other SOEs. For At-SO the low-pH form is produced [19], and for HSOR160Q a new form (species 2) is observed under these conditions [27].</p><p>To strengthen the assignments of the observed EPR-active species in SDHR55M and SDHR55Q to the high-pH and blocked forms, the ESEEM spectroscopic experiments described later were performed to directly establish the presence of OH or SO42− ligands.</p><!><p>On the basis of the principal g values, the Mo(V) species of SDHR55M is likely to represent the usual high-pH form, with an exchangeable equatorial OH ligand. The OH bond makes a large angle with the equatorial plane where the Mo(V) dxy orbital carrying the unpaired electron is located. This results in the isotropic hyperfine interaction constant, aiso, of the OH proton being close to zero and in the absence of hyperfine splittings in the CW EPR spectrum. In contrast, the anisotropic hyperfine interaction of this proton is fairly strong, T⊥ ~ 7–9 MHz [14, 18], and the detection of such a proton verifies the presence of the equatorial OH ligand.</p><p>ESEEM methods have proved to be of great utility for investigating the protons of the equatorial OH ligand of the high-pH form [14, 18]. The spectra obtained by some of the ESEEM techniques contain not only the fundamental nuclear transition lines at frequencies να and νβ, but also the sum and difference combination lines, νσ = να + νβ and νδ = |να − νβ|, respectively. For a nucleus of spin I = 1/2 in the case of weak hyperfine interaction (A < 2νI, where A is the total hyperfine interaction constant contributed by both isotropic hyperfine interaction and anisotropic hyperfine interaction, and νI is the nuclear Zeeman frequency) the sum combination line is located at the frequency [32]</p><p>Equation 1 allows one to estimate the anisotropic hyperfine interaction constant, T⊥, from the position of the sum combination line detected in an ESEEM spectrum. Although this equation was derived for the situation of complete orientational disorder, it is also approximately applicable to the ESEEM spectra obtained at the orientationally disordered EPR position gy.</p><p>The two-pulse ESEEM is the simplest technique to obtain spectra that contain the νσ line. However, this technique has two major drawbacks: a relatively low frequency resolution limited by the electron spin transverse relaxation; and a relatively large dead time (tens or hundreds of nanoseconds, depending on the microwave frequency band). The latter circumstance limits the observation possibilities for broad lines and results in characteristic oscillatory distortions in the cosine Fourier transform spectra phased to account for the dead time. An alternative technique that allows one to observe the sum combination feature and which is free from the above-mentioned drawbacks is the τ-integrated four-pulse technique [33, 34] (where τ is the time interval between the first and second microwave pulses), and this technique was used in this work to detect the 1H νσ line for the samples of SDH.</p><p>Figure 2 shows the 1H sum combination line region of the experimental Ku band τ-integrated four-pulse ESEEM spectra of SDHR55M prepared in H2O and D2O buffers (solid and dashed traces, respectively). The spectra were obtained at the intermediate EPR turning point corresponding to gy. The narrow negative peak located at exactly 2νH (νH is the 1H Zeeman frequency) represents the sum combination line due to distant protons, most of which belong to the protein and are not exchangeable. The broader negative peak that is located at a somewhat higher frequency and which is not present in the spectrum of the sample prepared in the D2O buffer arises from a neighboring exchangeable proton. The value of the shift Δνσ = νσ − 2νH ≈ 1.58 MHz gives an estimate of the anisotropic hyperfine interaction constant T⊥ & −8.7 MHz, quite typical for the hydroxyl ligand proton in high-pH SOEs [14, 18].</p><p>Identical results were obtained for the high-pH SDHR55Q enzyme, where the anisotropic hyperfine interaction constant was also estimated to be about −8.7 MHz. Thus, along with the observation of a characteristic set of principal g values by CW EPR, the ESEEM experiments establish that the Mo(V) signal of SDHR55M and the high-pH signal of SDHR55Q do indeed belong to the well-known high-pH form of SOEs.</p><!><p>The CW EPR results suggest that species 1 of SDHR55Q observed at low pH is a blocked form with coordinated sulfate. If this assumption is correct, then, similar to the previously studied blocked forms of SO [27, 30], the sulfur atom of the sulfate ligand should be observable by ESEEM for a sample prepared with 33S-enriched sulfite. Therefore, low-pH samples of SDHR55Q were prepared with sulfite having a natural abundance of sulfur isotopes (94.93% 32S, I = 0) and with 33S-enriched sulfite (99% 33S, I = 3/2). These samples will be referred to as 32S-SDHR55Q and 33S-SDHR55Q, respectively.</p><p>Figure 3 shows the Ka band two-pulse ESEEM spectra of 33S-SDHR55Q (solid traces) and 32S-SDHR55Q (dashed traces), obtained at the EPR turning points of species 1. The difference between the spectra of the two samples is caused by the interaction of the unpaired electron of Mo(V) with 33S. The results in Fig. 3 prove that species 1 of SDHR55Q is a blocked form of the enzyme, with bound sulfate, analogous to species 1 of HSOR160Q that was studied previously [27]. However, in contrast to the blocked forms of At-SO and HSO studied previously [27, 30] the blocked form of SDHR55Q shows the largest amplitude of the 33S ESEEM at the low-field and high-field EPR turning points (gz and gx, respectively). Toward the central parts of the spectrum the 33S ESEEM rapidly decreases, and near gy it is virtually unobservable. In the hyperfine sublevel correlation (HYSCORE) spectra of 33S-SDHR55Q (Fig. 4) the off-diagonal correlation lines were only seen at gz and gx, whereas the HYSCORE spectra obtained at the inner parts of the EPR spectrum only showed weak diagonal lines that might have originated from the contribution of a stimulated electron spin echo (ESE) signal to the HYSCORE ESE signal [35].</p><p>In our previous investigations of the blocked forms of SOEs we found that the NQI of the 33S nucleus is much stronger than the Zeeman (under the Ka band experimental conditions) and hyperfine interactions (e2Qq/h is approximately tens of megahertz, whereas νI ~ 3.5 MHz at Bo ~ 1 T, and A is approximately a few megahertz) [27, 30]. The ESEEM theory for such a spectroscopic situation was developed in our previous work [30]. According to that theory, the strong NQI splits the nuclear spin states of 33S into two doublets of degenerate |±3/2〉 and |±1/2〉 states (similar to Kramers doublets in EPR), whereas the Zeeman and hyperfine interactions lift the degeneracy. The transition frequency between the states that belong to different doublets (the interdoublet transition frequency) is</p><p>The most easily observable HYSCORE cross-peaks belong to |1/2〉 ↔|−1/2〉 transitions of 33S within the |α〉 and |β〉 electron spin manifolds. The frequencies of these transitions, να and νβ, are</p><p>
(3)vα,β≈chq(vI±A/2), where chq ∈ [1, 2 + η] is a numerical factor that depends on the orientation of the Bo vector relative to the NQI principal axes frame (η is the asymmetry parameter of the NQI tensor).</p><p>The HYSCORE spectrum obtained at gx is qualitatively similar to that obtained earlier for HSOR160Q, and it is the only spectrum that reveals an interdoublet transition(s) line at a frequency of νid ~ 13.7 MHz (Fig. 4, spectrum c). Using Eq. 2, one can estimate from this frequency the quadrupole coupling constant, e2Qq/h ~ 2νid ~ 27 MHz. From the positions of the correlations peaks at gz (4.6, 2.8) MHz and gx (8.5, 7.2), using Eq. 3, one can estimate Az ≈ 1.7 MHz and Ax ≈ 0.6 MHz. Thus, the 33S NQI and hyperfine interaction parameters for species 1 in SDHR55Q (at least those which could be determined) are in qualitative agreement with those reported for HSOR160Q [27]: e2Qq/h ≈ 36 MHz, Az ≈ 3.4 MHz, and Ax ~ 0 MHz.</p><p>The most likely reason for the observed rapid decrease of the 33S ESEEM amplitude in the central parts of the EPR spectrum is a static statistical distribution of the sulfate ligand orientations within certain (possibly rather narrow) limits. Such a distribution of orientations has been previously documented for the bound phosphate group in the phosphate-inhibited form of chicken SO [25]. An example of a structural model that could explain the relatively sharp 33S ESEEM spectral features at gx and gz and their virtual absence at gy is as follows. Suppose the long axis of the NQI tensor is approximately in the xz-plane of the g-frame, and the static structural distribution consists of the departures of the orientation of this axis from the xz-plane by a small angle θ. The projection of this axis on the g-frame axis y will change in proportion with θ. At the same time, the projections on x and z will change in proportion with θ2 ≪ θ. The changes of the y-projection will result in a distribution of chq factors in Eq. 3, and in a dramatic broadening and decrease in amplitude of the ESEEM spectral lines at the gy EPR position. A variation in the hyperfine interaction parameters caused by the structural distribution will also be a factor contributing to the line broadening and decrease of the ESEEM amplitude at gy.</p><!><p>As in earlier works with SOEs [36, 37], the experiments with samples prepared in a buffer containing H217O show an exchange of the axial oxo ligand with 17O. The 17O NQI constant e2Qq/h can be readily estimated from the ESEEM spectra containing the sum combination feature, νσ, if the NQI is weak compared with the nuclear Zeeman and hyperfine interactions: e2Qq/4I(2I − 1)h < νO ± A/2, where νO is the Zeeman frequency of 17O. The 17O nucleus has spin I = 5/2, and the sum combination feature represents a quintet with the splittings between the individual lines being determined by the NQI [38]. On the basis of the general expression derived elsewhere [38], the largest observable splitting δνσ translates to the quadrupole coupling constant as</p><p>The ESEEM experiments have shown that for SDHR55M the NQI splittings in the 17O νσ feature could only be partly resolved, even in the spectra obtained by the integrated four-pulse technique (which has high resolution limited by the electronic longitudinal relaxation time, T1), whereas for species 1 of SDHR55Q the splittings at gz could already be resolved by the two-pulse ESEEM (resolution limited by the transverse relaxation process). The corresponding spectra are shown in Fig. 5. The maximum estimated splitting for SDHR55M is about 0.22 MHz (e2Qq/h ~ 0.7 MHz), whereas in SDHR55Q the splittings are about 1.2 MHz (e2Qq/h ~ 4 MHz).</p><!><p>When studying the blocked form of the Mo(V) center of HSOR160Q, we observed ENDOR lines from a neighboring exchangeable proton. The anisotropic hyperfine interaction of this proton was significantly smaller than that of an equatorial OH ligand, but the hyperfine interaction tensor was rhombic, which is characteristic of a composite tensor with contributions from at least two centers of spin density localization. This tensor was therefore interpreted as indicative of a proton from a water molecule or an OH group that was hydrogen-bonded to the coordinated oxygen of the equatorial sulfate ligand [27]. The zero isotropic hyperfine interaction constant (aiso = 0 MHz) of this proton has excluded an alternative explanation that the spin density was delocalized on an orbital responsible for a covalent bond of the respective hydrogen atom with another atom.</p><p>Since it was established above that the low-pH Mo(V) center of SDHR55Q is in the blocked form, the possibility of a similar neighboring proton in SDHR55Q was also investigated. Figure 6 shows the 1H refocused Mims ENDOR spectra [39, 40] of the exchangeable protons obtained as a difference between the spectra of the samples prepared in H2O and D2O. The original spectra were recorded at the EPR turning points of the blocked form of SDHR55Q (species 1) and were normalized by the refocused stimulated ESE amplitude. For the gy turning point the minor (approximately 20%) contribution of the high-pH form was subtracted from the total ENDOR signal.</p><p>The spectra in Fig. 6 are virtually identical to the refocused Mims ENDOR spectra of exchangeable protons obtained previously for the blocked form of HSOR160Q [27], and very similar numerical simulation results for the hyperfine interaction parameters are obtained for SDHR55Q, where the average isotropic hyperfine interaction constant 〈aiso〉 = 0 MHz, and the anisotropic hyperfine interaction tensor T = (−3.2, −1.8, 5.0) MHz. The spectra simulated with these parameters are shown by dashed lines in Fig. 6.</p><!><p>The experiments with SDHR55M have revealed that only the high-pH form of the Mo(V) center is generated at various pH values, as occurs with SDHWT [21, 22]. On the other hand, for SDHR55Q the pH-dependent formation of two species was detected. One of the species is the usual high-pH form, which is almost exclusively present at pH 9. The second form, species 1, is the major species at pH < 7 and was shown to be the blocked form by reduction with 33S-enriched sulfite.</p><p>SDHR55Q is the second R → Q SOE active-site mutant studied by EPR. The CW EPR spectra and all of the high-resolution spectroscopic observations for the blocked form of SDHR55Q are similar to those for HSOR160Q reported previously [27]. Thus, the general structure of the blocked form of SDHR55Q should be similar to that proposed for HSOR160Q [27].</p><p>Another interesting observation for the R → Q mutants is that a typical low-pH EPR signal with large isotropic splittings from an exchangeable proton is never observed with sulfite reduction. SDHR55Q shows only the blocked and high-pH forms as a function of pH. HSOR160Q shows these signals and an additional low-pH EPR signal (species 2) of as yet unknown origin that can also be generated by reduction with Ti(III) [27]. For At-SO, reduction with sulfite at low pH produces the blocked form, but reduction with titanium(III) citrate at low pH generates a typical low-pH spectrum [19]. In contrast, reaction of SDHR55Q, with Ti(III) at pH 5.8 resulted only in the formation of small amounts of the high-pH form.</p><p>It is also interesting to compare the g values observed for the blocked forms of the SDHR55Q and HSOR160Q mutants with those observed for At-SOWT and HSOY343F. The values for the latter two are similar to those of low-pH SO (2.005, 1.974, and 1.963) [19, 23, 30], whereas the R → Q mutants exhibit a significant decrease in gx, from 1.963 down to 1.95 (Table 1). On the basis of simple crystal field considerations, such a change in gx can qualitatively be rationalized as resulting from a change in the energy of the dyz orbital, which again may represent the effect of the hypothesized axial coordination of the glutamine residue in the HSOR160Q mutant [27, 28].</p><p>One of the points of special interest for SOEs is the quadrupole coupling constant of the axial 17O-oxo ligand in the blocked forms. For both HSO and SDH, replacing the active-site arginine by a glutamine resulted in dramatic (fourfold to fivefold) increase of the quadrupole coupling constant of this 17O ligand compared with the values of e2Qq/h ≤ 1.5 MHz typically observed for other SOEs [38, 41], for model oxo-Mo(V) compounds, [13, 38, 41], and for SDHR55M in this work. In our previous work on HSOR160Q [27] we hypothesized that this effect could be caused by weak coordination of the carbonyl oxygen of glutamine in the axial position of the Mo(V) center, trans to the oxo ligand. This hypothesis was supported by the oxo-17O NQI parameters obtained for a six-coordinate model complex (dttd)Mo17O(17Otms), where H2dttd is 2,3:8,9-dibenzo-1,4,7,10-tetrathiadecane and tms is trimethylsilyl, e2Qq/h ≈ 3 MHz vs. 1.5 MHz in five-coordinate [Mo(V)17O(SPh)4]− [27]. A similar suggestion was made earlier by Doonan et al. [28] on the basis of the extended X-ray absorption fine structure data for HSOR160Q.</p><p>The high-pH form is remarkably similar for SOEs from all organisms [13], and no significant shifts in principal g values are observed among either wild-type or mutant sources (Table 1). These EPR data imply that at high pH the active-site R → Q mutations (HSOR160Q and SDHR55Q) do not significantly perturb the five-coordinate active-site structures observed by X-ray crystallography for the wild-type enzymes [6, 7] and confirmed by pulsed EPR spectroscopy for native chicken SO [18].</p><p>Finally, the spectroscopic results presented here also reinforce our previous structural [7, 11, 42] and kinetic [11, 12, 22, 43] studies of SDHWT and various R55 mutants. Earlier literature [10] has suggested that the mechanism of SOEs involves nucleophilic attack of sulfite on the oxo-Mo(VI) center to form the enzyme–substrate complex (k1 of Scheme 1), which then dissociates to give sulfate and Mo(IV) (k2). Two sequential one-electron oxidations of the molybdenum center (k4 and k7) regenerate Mo(VI). However, recently it has been proposed that one-electron oxidation to Mo(V) (k3) can precede product dissociation in SDH [12]. This hypothesis is supported by the observation of the blocked form of SDHR55Q by pulsed EPR studies in this work and by the previous observation that the one-electron intramolecular electron transfer rates between the molybdenum and heme centers for SDHWT, SDHR55M, and SDHR55Q are all much faster than kcat [12].</p><!><p>CW and pulsed EPR spectroscopy have been used to characterize two mutations of the conserved active-site arginine residue in bacterial SDH, R55M and R55Q. Both mutations replace the positively charged arginine by a neutral amino acid residue, but their effects are very different. Substitution by the neutral hydrophobic methionine residue (SDHR55M) had essentially no effect on the EPR properties of the Mo(V) center, even though the crystal structure of oxidized SDHR55M shows significant differences from the structure of SDHWT (Fig. 7) [11]. For example, the structure of SDHR55M has a sulfate bound at the active site, and the methionine side chain is bent away from the active site into a small cavity between the side chains of L121 and Q33. The space that was occupied by R55 in SDHWT appears to be largely empty in SDHR55M. No crystal structure is available for SDHR55Q, but the substitution of the positively charged arginine by the neutral hydrophilic glutamine residue (SDHR55Q) resulted in a dramatic change of the pH dependence of the EPR spectra compared with SDHWT. For SDHR55Q the blocked form is generated at low pH. Similar to the blocked form of HSOR160Q, the quadrupole coupling constant (e2Qq/h) of the presumed axial 17O-oxo ligand is fourfold to fivefold greater than the values typically observed for other SOEs [38, 41]. This effect has been hypothesized to result from weak coordination of the carbonyl oxygen of the substituted glutamine in the axial position of the Mo(V) center, trans to the oxo ligand [27, 28]. However, unlike HSOR160Q, the SDHR55Q mutant does not show an additional EPR form at intermediate pH (approximately 7), and the high-pH form dominates at pH ≥ 8. The reasons for this difference between HSOR160Q and SDHR55Q, as well as the origin of species 2 observed for HSOR160Q at pH ~ 7, are not currently clear. EPR experiments on the structure of species 2 of HSOR160Q are in progress and may help resolve these differences between the SDH and HSO systems.</p><!><p>Continuous-wave electron paramagnetic resonance (EPR) spectra of sulfite-reduced SDHR55M at pH 9.0 (trace 1) and pH 5.8 (trace 2), and sulfite-reduced SDHR55Q at pH 9.0 (trace 3) and at pH 5.8 (trace 4). The experimental conditions were as follows: νmw = 9.450 GHz; microwave power, 2 mW; modulation amplitude, 0.2 mT; temperature, 77 K. The EPR turning points for the high-pH-like form are shown by asterisks. The turning points for species 1 of SDHR55Q are shown by diamonds</p><p>1H sum combination line region of cosine Fourier transform (FT) spectra of τ-integrated four-pulse electron spin echo envelope modulation (ESEEM) of the Mo(V) center of SDHR55M (pH 5.8) prepared in H2O (solid trace) and D2O (dashed trace). The experimental conditions were as follows: νmw = 17.362 GHz; Bo = 635.5 mT (gy); microwave pulses, 6, 6, 13, and 6 ns; temperature, 21 K</p><p>Amplitude FT spectra of normalized two-pulse ESEEM obtained for low-pH (pH 5.8) samples of 33S-SDHR55Q (solid lines) and 32S-SDHR55Q (dashed lines), respectively. The spectra were obtained at the EPR turning points of species 1 and have the same vertical scale. a, b, and c correspond to gz, gy, and gx, respectively. The 33S lines are clearly seen at gz and gx positions, but virtually disappear at gy. The experimental conditions were as follows: νmw = 29.568 GHz; Bo = 1,057.9 mT (gz), 1,078.4 mT (gy), and 1,088.4 mT (gx); microwave pulses, two of 16 ns; temperature, 21 K</p><p>33S hyperfine sublevel correlation spectra of low-pH (pH 5.8) samples of 33S-SDHR55Q obtained at the EPR turning points of species 1. a, b, and c correspond to gz, gy, and gx, respectively. The spectra were normalized by the amplitudes of the stimulated electron spin echo signal at the measurement positions. The relative scales are ×1 for a and c, and ×2 for b. The experimental conditions were as follows: νmw = 29.568 GHz; Bo = 1,057.9 mT (gz), 1,078.4 mT (gy), and 1,088.4 mT (gx); microwave pulses, 14, 14, 25 and 14 ns; temperature, 21 K. Each spectrum represents a sum of the spectra obtained at the time intervals τ between the first and second microwave pulses of 170 and 200 ns</p><p>Cosine FT spectra of τ-integrated four-pulse ESEEM of 17O-SDHR55M (trace 1) and two-pulse ESEEM of 17O-SDHR55Q, species 1 (trace 2) obtained at the respective low-field EPR turning points (gz). The stick diagram over each spectrum indicates the sum combination (νσ) feature split into a quintet by the 17O nuclear quadrupole interaction. The experimental conditions for trace 1 were as follows: νmw = 29.640 GHz; Bo = 1,070 mT (gz); microwave pulses, 11, 11, 21 and 11 ns; temperature, 21 K. The experimental conditions for trace 2 were as follows: νmw = 29.592 GHz; Bo = 1,058.6 mT (gz); microwave pulses, two of 15 ns; temperature, 21 K</p><p>Solid traces are the 1H electron–nuclear double resonance (ENDOR) spectra at the EPR turning points (traces 1–3) obtained as a difference between the refocused Mims 1H ENDOR spectra recorded for species 1 of SDHR55Q in H2O and D2O and normalized by the refocused stimulated electron spin echo amplitude. The experimental conditions were as follows: Bo = 1,057.5 mT (gz), 1,077.4 mT (gy), and 1,087 mT (gx) for traces 1, 2, and 3, respectively; νmw = 29.555 GHz; microwave pulses, three of 15 ns and one of 22 ns; time interval between the first and second microwave pulses, τ = 80 ns; time interval between the second and third microwave pulses, T = 30 μs; time interval between the third and fourth (refocusing) microwave pulses, τ′ = 380 ns; RF pulse length, 20 μs; temperature, 21 K. Dashed lines are numerical simulations for 〈aiso〉 = 0; Δaiso = 0.5 MHz; T = (−3.2, −1.8, 5.0) MHz; (ϕh, θh, ψh) = (70°, 80°, 0°)</p><p>Superimposed structures of SDHWT (blue arginine residue) and SDHR55M (red methionine residue), adapted from Protein Data Bank entries 2bpb and 2ca3</p><p>The sequence of steps which transform the enzyme–substrate complex, MoIV(OSO32−), into product (sulfate) and the reoxidized Mo(VI) state of the enzyme. In the pathway colored red, product release precedes reoxidation of the enzyme. For the pathway colored blue, the enzyme–substrate complex is oxidized by one electron prior to product release, and the paramagnetic blocked form, MoV(OSO32−), can be detected by electron paramagnetic resonance spectroscopy [12]</p><p>Principal g values of the Mo(V) center in SDHR55M and SDHR55Q</p><p>The g values for other sulfite-oxidizing enzymes are given for comparison</p><p>SDH sulfite dehydrogenase, At-SO sulfite oxidase from Arabidopsis thaliana, HSO human sulfite oxidase</p><p>The accuracy is ±0.0005</p>