Structures and reaction pathways of the molybdenum centres of sulfite-oxidizing enzymes by pulsed EPR spectroscopy
SOEs (sulfite-oxidizing enzymes) are physiologically vital and occur in all forms of life. During the catalytic cycle, the five-co-ordinate square pyramidal oxo-molybdenum active site passes through the Mo(V) state, and intimate details of the structure can be obtained from variable frequency pulsed EPR spectroscopy through the hyperfine and nuclear quadrupole interactions of nearby magnetic nuclei. By employing variable spectrometer operational frequencies, it is possible to optimize the measurement conditions for difficult quadrupolar nuclei of interest (e.g. 17O, 33S, 35Cl and 37Cl) and to simplify the interpretation of the spectra. Isotopically labelled model Mo(V) compounds provide further insight into the electronic and geometric structures and chemical reactions of the enzymes. Recently, blocked forms of SOEs having co-ordinated sulfate, the reaction product, were detected using 33S (I = 3/2) labelling. This blocking of product release is a possible contributor to fatal human sulfite oxidase deficiency in young children.
Introduction<!>Axial oxo-17O ligand<!>Detection of a nearby Cl- in lpH SOE<!>Using the 33S labelling to identify the blocked forms<!>Conclusions
<p>SOEs (sulfite-oxidizing enzymes) catalyse the oxidation of sulfite to sulfate as the terminal step in the metabolism of sulfur-containing amino acids, and they are physiologically vital for all forms of life. The crystal structures of SOEs [1-3] from vertebrates, plants and bacteria show nearly identical, approximately square-pyramidal, five-coordinate geometries of their molybdenum centres in the fully oxidized Mo(VI) state, with axial and equatorial oxoligands, and three equatorial sulfur ligands (two from the pyranopterindithiolate cofactor and one from a cysteinyl side chain). In the first step of the proposed catalytic cycle [4] (Figure 1, A→B), the equatorial oxo ligand reacts with sulfite to form an enzyme-product complex, in which sulfate is co-ordinated to Mo(IV). Replacement of the sulfate ligand by water or hydroxide (Figure 1, B→C) and subsequent one-electron oxidations [Mo(IV/V/VI)] and concomitant deprotonations (Figure 1, C→D→A)return the molybdenum centre to the fully oxidized resting state. The paramagnetic Mo(V) state (Figure 1, D) is relatively stable, and EPR spectroscopy has provided a convenient method of directly investigating the structures of various Mo(V) species as a function of pH, anions in the medium, organism, method of generation of the Mo(V) state and enzyme mutations.</p><p>Historically, Bray and co-workers [5-7] first classified vertebrate SOEs into three forms based upon the appearance of their CW (continuous wave) EPR spectra: the high-pH form (hpH), generated at pH ≥9; the low-pH form (lpH) generated at pH ≤7.5 (depending on the concentration of Cl- in the buffer); and the phosphate-inhibited form (Pi)that was observed at low pH in the presence of phosphate. The CW EPR spectra provided a convenient phenomenological classification of SOEs, but the spectral resolution was too low to define the structural details of the Mo(V) centres generated under different conditions or, in some cases (e.g. for the hpH and Pi SOE), even to help identify the nature of the exchangeable equatorial ligand. Detailed structural investigations of the Mo(V) centres of SOEs require higher-resolution techniques, and ESEEM (electron spin-echo envelope modulation) and ENDOR (electron nuclear double resonance) spectroscopies, supplemented by the extensive isotopic labelling and site-directed mutagenesis, have become the methods of choice for such investigations.</p><p>In recent years, pulsed EPR experiments have revealed a wealth of information about the structural details for the 'original' three forms of SOE introduced by Bray [8]. Additional investigations have demonstrated that, under certain conditions, reduction with sulfite leads to formation of a Mo(V) centre in which the reaction product (sulfate) remains co-ordinated. This new form was called blocked (Figure 1) because it was hypothesized that the sulfate ligand was prevented from leaving the active site (i.e. blocked) by some steric hindrance [9].</p><p>The blocked form (Figure 1) was first identified for plant SOE from Arabidopsis thaliana (At-SO) by observing the 33S-ESEEM in samples prepared with 33S-labelled sulfite at low pH [9]. A similar method was used later to detect blocked forms for some of the mutants of human sulfite oxidase (hSO) [10]. In addition, the pulsed EPR results for the R160Q mutant of hSO [10] supported the suggestion that, in some of these blocked forms, the Mo(V) centre can be six-co-ordinate, with an additional (axial) ligand trans to the oxo ligand [11].</p><p>Within the EPR spectroscopic framework, the structural information about the SOEs is obtained from the hyperfine interaction (hfi) and nuclear quadrupole interaction (nqi) parameters of magnetic nuclei in the vicinity of the Mo(V) centre. The complexity of estimating the hfi and nqi parameters depends upon the type of magnetic nucleus. For example, the detection of 1H and 2H and analysis of their spectra are rather straightforward. On the other hand, the pulsed EPR investigations of 17O, 33S, 35Cl and 37Cl are currently in an embryonic stage, and many results are from our laboratory. In an Mo(V) site, these quadrupolar nuclei often have a combination of spectroscopic parameters that is unfavourable for easy detection and/or spectral interpretation. To enable their successful investigation, the level of theory should allow qualitative estimates of the hfi and nqi parameters to be obtained directly from the spectra. For detecting these quadrupolar nuclei, it is of a paramount importance that the experiments be performed at appropriate operational microwave frequencies, νmw, specially selected to optimize the detection and/or the analysis of the spectra for the nucleus of interest. Below we present some recent applications of variable-frequency ESEEM spectroscopy to the study of such difficult nuclei in the vicinity of the Mo(V) centres of SOEs.</p><!><p>There are two oxygen ligands at the Mo(V) centre (see D in Figure 1), one of which is the oxo ligand, and the other one is the oxygen of an equatorial OH ligand. The main oxygen isotope (16O, 99.76%) has spin I = 0, and, to be observable by EPR, oxygen has to be enriched in 17O (I = 5/2). Early CW EPR experiments in 17O-enriched water revealed the hyperfine splittings as large as ~35 MHz that were assigned to the equatorial OH ligand [6,12]. More recent pulsed EPR investigations in our laboratory have shown that the axial oxo ligand also exchanges in H217O [13]. The hfi of the oxo-17O was found to be substantially smaller, which is why it was not resolved by CW EPR. Importantly, the pulsed techniques also allowed determination of the nqi, which cannot, in principle, be measured directly by CW EPR.</p><p>The simple theory behind these measurements is as follows. When the hfi and nqi are weak compared with the nuclear Zeeman interaction, the frequencies of the nuclear transitions between the spin projections mI and mI + 1 (mI ≡ 〈IZ〉) within the α and β electron spin manifolds [14,15] are: να,β≈νI±12(aiso+TZZ)+TZX2+TZY28νI+Q~(2mI+1) where νI is the nuclear Zeeman frequency, aiso is the isotropic hfi constant, Tij are the components of the anisotropic hfi tensor and Q~∝e2Qq∕h is the nqi-related parameter. The first-order hfi term, aiso + TZZ, can be measured as να(mI) - νβ(mI). Although the nqi splits each of the fundamental frequencies (να and νβ) into 2I different components, these splittings are usually impossible to detect in one-dimensional experiments because of the broadening of the fundamental lines induced by the anisotropic hfi. Fortunately, however, in addition to the fundamental lines, the spectra obtained by some of the ESEEM techniques also contain a sum combination line at the frequency νσ = να + νβ. For a nucleus of spin I, in the limit of weak hfi and nqi, the sum combination line splits into a multiplet of 2I lines with the frequencies: νσ(mI)≈2νI+TZX2+TZY24νI+2Q~(2mI+1)</p><p>The hfi term causes the second-order frequency shifts that are useful for estimating the anisotropic hfi, but may be detrimental for the nqi estimates. However, it can be made arbitrarily small by increasing νI through an increase in the resonance magnetic field, Bo, concomitant to the increase in the microwave operational frequency. The splitting between any two neighbouring νσ lines is determined only by the nqi: Δνσ=∣νσ(mI+1)−νσ(mI)∣≈4Q~</p><p>Examples of spectra of axial 17O in model Mo(V) complexes and SOEs obtained from various organisms and mutants, detected by the τ -integrated four-pulse ESEEM technique, are shown in Figure 2. The 17O hfi obtained from the positions of the fundamental lines is 5-6 MHz for all varieties of SOE and model systems examined. Evidently, this 17O hfi value is characteristic of an oxo group (Mo ≡ O entity). The nuclear quadrupole coupling constant, e2Qq/h, determined from the splitting between the components of the νσ quintet was under 1.5 MHz for a five-co-ordinate model compound (Figure 2, trace 1) and for all SOEs investigated (Figure 2, traces 3-5) except the R160Q mutant of hSO (trace 6, Figure 2). The similar values of e2Qq/h imply that no drastic changes in the geometry of the Mo(V) centre occur among organisms and with varying pH.</p><p>In the R160Q mutant of hSO, the mutation of the active-site Arg160 [located on the side of the Mo(V) centre opposite to the oxo-ligand] to a glutamine residue has resulted in the dramatic increase of the oxo-17O e2Qq/h to approx. 5 MHz (Figure 2, trace 6) [10]. We hypothesized that this increase in the nqi could result from co-ordination of the active-site Glu160 to the Mo(V) centre trans to the oxo-ligand [10]. This hypothesis is in line with the earlier EXAFS results [11], and is supported by the observation of significant increase in the oxo-17O nqi in a model six-co-ordinate Mo(V) complex (Figure 2, trace 2) as compared with the five-coordinate one (~3 MHz compared with 1.45 MHz) [10,14]. From these results, it appears that the oxo-17O nqi provides a simple and dependable means to follow the co-ordination changes of the Mo(V) site of SOEs.</p><!><p>On the basis of the pH-dependence of the CW EPR of vertebrate SOEs in the presence of different concentrations of Cl-, Bray et al. [7] proposed that Cl- should be an integral part of the Mo(V) active centre in the lpH SOE. However, they did not succeed in confirming its existence directly. Recently, the quest to find this form was continued [16], but, despite considerable effort, these recent CW EPR experiments still do not unambiguously demonstrate coordination of Cl- to Mo(V).</p><p>The way to clearly prove co-ordination of Cl- to Mo(V) is by ESEEM spectroscopy. Indeed, the experiments on the lpH chicken SO (sulfite oxidase) with the natural abundance of the Cl isotopes and on samples enriched in 35Cl and 37Cl (both I = 3/2) have shown unequivocally the presence of Cl- in vicinity of the Mo(V) centre. From the hfi and nqi parameters (aiso ~4-5 MHz; T⊥ ~0.2 MHz; e2Qq/h ~3-4MHz for 35Cl), it is likely that this Cl- is weakly co-ordinated to the Mo(V) centre. From comparison with the model oxomolybdenum complexes with equatorial chloro ligands [17] and from the available general information on the structure of the Mo(V) centres of SOEs, it is clear that the co-ordination position of this weak chloro ligand should be axial, trans to the oxo ligand [18].</p><p>No 35Cl/37Cl ESEEM was detected for hpH SOE or for the blocked forms of SOE. On the other hand, lpH SOE prepared without added Cl- still shows Cl ESEEM with the same amplitude as lpH samples containing ~100 mM Cl-. These ESEEM results suggest a Cl--binding site of relatively high affinity. Cl- may play a physiological role in fine tuning the pKa of the OH ligand of the Mo(V) centre, which is observed to shift by ~1-1.3 pKa units for lpH SOE with added Cl- [7,16]. Using the analysis of the effect of a unit charge on the pKa of a titratable group [19,20], gives an estimate of ~4-4.5Å (1Å = 0.1 nm) for the distance from the Cl- ion to the OH ligand.</p><!><p>In contrast with vertebrate SOEs, the Mo(V) centre of At-SO did not reveal the presence of nearby exchangeable protons upon reduction with sulfite at low pH [21]. Therefore we proposed that the At-SO protein at low pH adopts a 'closed' conformation that forces the sulfate to remain co-ordinated to Mo(V) (see Figure 1), and the corresponding form of SOE was called blocked. To verify this hypothesis, At-SO was reduced with sulfite labelled with 33S (I = 3/2). As an example, in Figure 3(a), the field-integrated primary ESEEM spectrum of [33S]At-SO is compared with that obtained with the natural abundance of sulfur isotopes (94.93% 32S, I = 0). Figure 3(b) shows the HYSCORE (hyperfine sublevel correlation) spectrum for [33S]At-SO obtained at the low-field (gz) turning point of the EPR spectrum. The measurements were performed at νmw ~29 GHz which was estimated to be about the optimum microwave frequency for observing the co-ordinated sulfate [9].</p><p>The spectra of Figure 3 unambiguously demonstrate the co-ordination of sulfate to Mo(V). However, the evaluation of the spectroscopic parameters from these spectra was not straightforward because the 33S nqi was much stronger than the hfi and the Zeeman interaction. Such a case has been little analysed in ESEEM spectroscopy. To overcome this problem, we derived analytical expressions which allowed us to qualitatively interpret the ESEEM spectra and to estimate the interaction parameters from them [9,10]. In the present paper, we show some simplified results that are useful for qualitative understanding of the primary ESEEM and HYSCORE spectra.</p><p>A strong nqi of 33S separates its nuclear states into two degenerate pairs (doublets) with mI = ±1/2 and mI = ±3/2. The degeneracy is then removed by the nuclear Zeeman and hyperfine interactions. In the two-pulse ESEEM spectrum of Figure 3(a), the feature of interest is the peak at~18 MHz that belongs to one of the inter-doublet transitions (∣±1∕2〉↔∣±3∕2〉) of 33S within the electron spin manifold where the Zeeman and hyperfine interactions approximately cancel each other. The frequency of this inter-doublet transition is: νid∼e2Qq∕2h and we can easily estimate the quadrupole coupling constant as e2Qq/h≈36 MHz.</p><p>In the HYSCORE spectrum of Figure 3(b), the major off-diagonal cross-peaks at (3,11) MHz belong to ∣1∕2〉↔∣−1∕2〉 transitions of 33S. The frequencies of these transitions, να and νβ, are equal to: υαβ≈chq(υI±A∕2) where chq is a numerical factor that depends on the orientation of the magnetic field vector, Bo, relative to the nqi principal axes frame. The main qualitative difference with the I=1/2 system is that the pairs of HYSCORE cross-peaks are symmetrical with respect to νc = (να + νβ)/2 instead of νI.</p><p>Using these results, one can estimate the hfi constant as A = 2νI · (νβ - να)/(νβ + να). This estimate can then be refined by using numerical simulations of the HYSCORE spectra. Using this approach for [33S]At-SO has resulted in aiso =3.3 MHz; the anisotropic hfi tensor in the principal axes system (T11, T22, T33) = (1.3, 1.5, -2.8) MHz; e2Qq/h = 40 MHz and the nqi asymmetry parameter was found to be close to zero, η<0.3 (near-axial nqi tensor).</p><p>Further investigations have shown that the blocked form is not unique to At-SO: such forms have also been found for the R160Q and Y343F mutants of hSO [10], and recently, for the R55Q mutant of bacterial succinate dehydrogenase (analogous to R160Q hSO) (A.V. Atashkin, T. Rapson, U. Kappler and J.H. Enemark, unpublished work). For R160Q, the blocked form was present in significant quantities at physiological pH. Since this form represents a dead end in the catalytic cycle of Figure 1, this form may be a contributing factor in the lethality of the R160Q mutation in children.</p><!><p>The results presented above demonstrate the potential of high-resolution pulsed EPR techniques, particularly ESEEM spectroscopy of difficult quadrupolar nuclei, for structural investigations. The most important aspects of such investigations are the ability to freely adjust the microwave operational frequency to the needs of the particular spin system (in order to optimize the observation conditions for a particular magnetic nucleus and/or facilitate the interpretation of the ESEEM spectra) and selective isotopic labelling to make the nucleus of interest observable by ESEEM (e.g. 17O compared with 16O) or simply modify its spectroscopic parameters (e.g. 35Cl compared with 37Cl). The results presented here demonstrate applications to the Mo(V) centre of SOEs, but the same methods can be used for paramagnetic active sites of other types of metalloenzymes.</p>