Active site structures of the E. coli N-hydroxylaminopurine resistance molybdoenzyme YcbX

Abstract

X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) data have been used to characterize the coordination environment for the catalytic Mo site of E.coli YcbX in two different oxidation states. In the oxidized state, the Mo(VI) ion is coordinated by two terminal oxo ligands, a thiolate S from cysteine, and two sulfur donors from the bidentate pyranopterin ene-1,2-dithiolate (pyranopterin dithiolene; PDT). Upon reduction, it is the more basic equatorial oxo ligand that is protonated, with a Mo-O_eq bond distance that is best described as either a short Mo⁴⁺-OH₂ bond or a long Mo⁴⁺-OH bond. Mechanistic implications for substrate reduction are discussed in light of these structural details.

Graphical Abstract

Pyranopterin molybdenum enzymes are typically characterized as belonging to either the sulfite oxidase (SO), xanthine oxidase (XO), or dimethyl sulfoxide reductase (DMSOR) enzyme families.^1–5 However, the molybdenum cofactor sulfurase C-terminal (MOSC) domain enzymes^6–11 represent a new family of molybdenum enzymes that do not possess any detectable homology with the original three canonical molybdenum enzyme families, although the MOSC domain proteins appear to possess active site structures that are reminiscent of SO family enzymes. However, a key difference between MOSC domain enzymes and SO family enzymes is that the latter frequently fuse to cytochromes and do not have the [2Fe-2S] ferredoxin domains commonly seen in either MOSC (e.g., the bacterial YcbX and YiiM proteins) or XO family enzymes. The YcbX MOSC enzymes¹² are of intense interest since they are orthologues of the eukaryotic mitochondrial amidoxime reducing component (mARC) enzymes,^{6–8, 13–20} which are mostly represented by two paralogues, mARC1 and mARC2. The mARCs are biotransformation and moonlighting enzymes^{6, 13} that can reduce highly toxic or mutagenic N-oxygenated metabolites (Figure 1), which derive from the catalytic action of cytochrome P450s or flavin dependent monooxy-genases. Although the exact biological roles for mARC enzymes remain undetermined, YcbX and YiiM catalyze the reduction of N-hydroxylated purines to protect E. coli from the mutagenic and toxic effects of these compounds.^{12, 21} Catalytic electrochemistry studies show that YcbX is specific for N-hydroxylated substrates and does not catalytically reduce N-oxides.²² This substrate specificity indicates important differences in active site structure relative to other pyranopterin Mo enzymes, contributing to their respective reactivity profiles.

Figure 1.

Proposed mechanism of substrate reduction by MOSC family enzymes, including YcbX. Note that the Mo(VI) → Mo(IV) reduction step is expected to proceed via an obligatory Mo(V) intermediate.

The eukaryotic mARC enzymes are small monomeric proteins that do not contain prosthetic groups other than the pyranopterin dithiolene (PDT, also known as molybdopterin; MPT), but their bacterial counterparts are multidomain proteins with additional redox-active prosthetic groups. For example, E. coli YcbX binds both Mo-(MPT) and a [2Fe2S] cluster in addition to forming homodimers. Unfortunately, no crystal structure of E. coli YcbX has been published and no spectroscopic data on the molybdenum active site are available, further complicating the development of structure-property relationships among MOSC family enzymes. The only MOSC domain protein crystal structures are those of human mARC1⁷ and its disease-related p.A165T variant.²³ However, in these structures there is only partial occupancy of the Mo ion and the exact coordination environment about the Mo ion is not clearly defined. The structure of plant mARC1 in the oxidized Mo(VI) state has been interrogated by X-ray absorption spectroscopy (XAS).⁸ Using extended X-ray absorption fine structure (EXAFS), the active site coordination environment was observed to be similar to that of SO with the Mo ion being coordinated by the two S atoms of the PDT, a S atom from a conserved cysteine thiolate, and two terminal oxo ligands.⁸ Interestingly, there is no detectable similarity between the amino acid sequences or the protein folds of these MOSC enzymes and SO, despite their apparent active site structural similarity.

Although MOSC domain proteins are very highly abundant in nature, we know remarkably little about their protein and Mo active site structures. Here, we examine the first coordination sphere environment of the catalytic Mo ion in YcbX as a function of oxidation state and sample preparation using X-ray absorption near-edge structure (XANES) spectroscopy and EXAFS. The Mo K-edge XANES spectra for as-isolated, excess substrate oxidized, and dithionite-reduced E. coli YcbX are presented in Figure 2. The rising edge energies, determined by using the first derivative inflection point, are observed at 20016.2 eV (oxidized), 20015.4 eV (as-isolated), and 20012.5 eV (reduced) and these energies reflect Mo oxidation state and effective nuclear charge differences between the different enzyme forms. Further inspection of the data in Figure 2 shows that the as-isolated (Figure 2, blue) and excess substrate oxidized (Figure 2, red) enzyme XANES are slightly different. The fully oxidized Mo(VI) enzyme sample was obtained from the as-isolated protein by reacting it with excess substrate, benzamidoxime. Enzyme that has been reduced with dithionite displays a markedly different XANES spectrum (Figure 2, green). All three of the YcbX XANES spectra shown in Figure 2 display a typical “oxo-edge” transition in the pre-edge region at 20004.5 eV (oxidized), 20003.7 eV (as-isolated), and 20003.3 eV (reduced). The intensity of this “oxo-edge” pre-edge feature can be correlated with the number of terminal oxo ligands that are covalently bound to the metal ion.^{8, 24–26} Thus, the increased intensity of this feature in the oxidized and as-isolated data sets clearly indicates a greater number of terminal oxo ligands bound to Mo in these enzyme forms when compared to the reduced enzyme form.

Figure 2.

Mo K-edge XANES spectra for E. coli YcbX as a function of oxidation state and sample preparation.

The EXAFS data (k-range 3-12 Å⁻¹) for oxidized and reduced YcbX are presented in Figure 3 and summarized in Table S1. The data clearly indicate that both O_oxo and S donor shells dominate the first coordination sphere of the Mo ion in the YcbX active site. For the oxidized species, the best fit to the EXAFS data yields a di-oxo site with Mo⁶⁺-O_oxo bond distances of 1.730 Å and three sulfur donors with Mo⁶⁺-S bond distances of 2.445 Å. These bond distances are very similar to those determined for the oxidized active sites of sulfite oxidase family enzymes^27–30 and plant mARC1⁸ (pmARC1). For the reduced YcbX enzyme form, the Mo(IV) ion is found to be coordinated by a single terminal oxo ligand (Mo⁴⁺-O_oxo = 1.707 Å), three S donors with Mo⁴⁺-S bond distances of 2.406 Å, and a non-oxo light atom at 2.194 Å. To investigate the possibility that S_cys has de-coordinated from the Mo(IV) site, we performed a bond valence sum (BVS) analysis^{31, 32} using the reduced YcbX EXAFS data. This analysis yields a BVS of 3.96, which is in excellent agreement with the Mo(IV) oxidation state assignment and is fully consistent with S_Cys remaining coordinated to Mo in reduced enzyme. The non-oxo light atom donor is generally accepted to be a water molecule in reduced forms of SO,²⁸ plant nitrate reductase (pNR),³³ and the C-terminal domain of the MOSC protein human molybdenum cofactor sulfurase (HMCS-CT).⁸ In these enzymes, the EXAFS determined non-oxo Mo⁴⁺-O bond distances vary by 0.31Å (2.30 Å, SO;²⁸ 2.18 Å, pNR;³³ 2.49 Å, HMCS-CT⁸). The non-oxo bond distance in XO is markedly shorter³⁴ at 1.98 Å and has been assigned as arising from Mo⁴⁺-OH.³⁵ DFT results for computational models of reduced SO result in 2.08 Å and 2.34 Å bond lengths for Mo⁴⁺-OH and Mo⁴⁺-OH₂, respectively.²⁸ However, there is some degree of uncertainty in these non-oxo Mo⁴⁺-O bond lengths due to the partially in-phase relationship between the weaker Mo⁴⁺-O EXAFS oscillations and the markedly more intense Mo⁴⁺-S EXAFS.³⁴ Thus, given the 2.194 Å Mo⁴⁺-O bond length determined for reduced YcbX, we attribute this to be either a short Mo⁴⁺-OH₂ bond or a long Mo⁴⁺-OH bond.³⁴ The crystal structure of the related human mARC1 shows that this equatorial ligand is oriented directly toward the solvent, and is therefore likely involved in catalysis.⁷ If the active site structure of reduced YcbX is [(PDT)Mo^IVO(S_Cys)(OH)]²⁻, with a hydroxide ligand coordinated to Mo(IV), this would have interesting consequences for the mechanism of substrate reduction since the coordinated hydroxide would have to be protonated, perhaps by the substrate, to allow for water labilization and binding of the activated anionic substrate (Figure 1).

Figure 3.

Mo K-edge EXAFS data for oxidized (A, D), as-isolated (B, E), and reduced (C, F) E. coli YcbX.

Close inspection of the EXAFS oscillations for the as-isolated enzyme (Figure 3, B and E) indicates that this preparation closely resembles the oxidized form (Figure 3, A and D). The slight differences between the oxidized and as-isolated samples can be explained by as-isolated being a mixture of oxidized and reduced forms. This is supported by the presence of the more pronounced EXAFS feature at ~4 Å⁻¹ in as-isolated, which clearly shows that some of the reduced enzyme form is also present. We have used a linear combination fit to show that the as-isolated sample is a mixture comprised of 88.5% oxidized and 11.5% reduced enzyme forms (Figure S1). Thus, the heterogeneity we observe in the as-isolated enzyme does not appear to derive from an inhibited or otherwise catalytically inactive species, indicating that the as-isolated enzyme is fully active. The observation that as-isolated YcbX is a mixture of oxidized and reduced enzyme forms is of interest, since pyranopterin Mo enzymes are usually obtained in the fully oxidized Mo(VI) state after aerobic purification.^{27, 36, 37}

Regarding the active site heterogeneity observed for SO family enzymes, EXAFS has been used to show that plant nitrate reductase (pNR) possess two different Mo(VI) forms.³³ Although the EXAFS data for oxidized as-isolated enzyme and as-isolated enzyme incubated with excess nitrite or nitrate are virtually identical, the addition of nitrate to reduced pNR leads to changes in the Fourier transform intensity ratios of the Mo≡O and Mo-S EXAFS peaks. Analysis of the EXAFS data indicates one of the Mo-S^ditholene bonds is elongated in oxidized as-isolated enzyme, with the nitrate-oxidized species possessing a catalytically relevant [(PDT)MoO₂(S_Cys)]¹⁻ active site with three equivalent Mo-S bonds. Interestingly, the nitrate-oxidized form reverts to the as-isolated form following gel filtration.³³ For the related SO family protein MsrP (formally known as YedY) the as-isolated form is an inhibited Mo(V) species with an equatorial thiol ligand replacing the H₂O/OH⁻.³⁸ In marked contrast, the active site heterogeneity that we observe here for YcbX is completely unrelated to these cases since simple incubation of as-isolated enzyme with substrate leads to a homogeneous Mo(VI) oxidized enzyme form. Furthermore, reduction with dithionite produces a similarly homogeneous reduced Mo(IV) species. The as-isolated protein does not contain any relevant Mo(V) species that are detectable by EPR spectroscopy. Thus, our enzyme preparations appear to contain intact, fully active protein in a mixture of two oxidation states. It is assumed that recombinant YcbX is reduced by its electron transfer partner CysJ in E. coli cells. In absence of substrate, the enzyme remains in this state due to the low redox potential within the E. coli cytosol. We hypothesize that when the cells are lysed and the protein isolated, the enzyme is apparently not completely oxidized by air, leading to the mixed Mo(IV)/Mo(VI) as-isolated state.

In summary, this is the first study in which the active site Mo coordination environment of a MOSC domain protein has been examined by EXAFS in both its reduced Mo(IV) and oxidized Mo(VI) forms for direct comparison with canonical SO family enzymes. While the structure of the YcbX Mo(VI) state is very similar to that of SO^39–43 and plant nitrate reductase (pNR),⁴⁴ there may exist subtle but important aspects of the Mo coordination sphere that contribute to their different reactivities.^{2, 45 , 46} Although there is no definitive experimental evidence for a direct oxygen atom transfer mechanism in SO or pNR,¹ this is the generally accepted mechanism and is supported by spectroscopic⁴⁷ and reaction coordinate computations.^{2, 14, 48–50} Key to this reactivity in SO is the electronic structure-induced activation of the equatorial oxo ligand for bond scission upon two-electron transfer from the substrate to a Mo=O_oxo d-p π* orbital.⁴⁷ The mechanism for pNR is proposed to be the reverse of that in SO, requiring the loss of a labile water ligand in reduced pNR to allow for direct binding of nitrate to Mo(IV) to initiate the oxygen atom transfer reaction. A thermodynamic basis for the differences in SO and pNR reactivity is related to the relative bond enthalpies for the substrate S=O and N=O bonds, and the Mo=O/Mo≡O bonds of the catalytic site.⁵¹ Additional studies of the reduced Mo(IV) state in YcbX are ongoing in order to confirm the nature of the second oxygen ligand as either a water or hydroxide, with a key emphasis on how this ligand and the nature of bound substrate contribute to the mechanism of substrate reduction by YcbX and related mARC family enzymes.

SYNOPSIS TOC.

X-ray absorption spectroscopy has been used to structurally characterize the oxidized and reduced forms of the MOlybdenum Sulfurase C-terminal (MOSC) domain protein YcbX. In the oxidized state, the Mo(VI) ion is coordinated by two terminal oxo ligands, a thiolate S from cysteine, and two sulfur donors from the pyranopterin dithiolene (PDT) ligand. In the reduced form of the enzyme, the Mo-Oeq bond distance is best described as either a short Mo⁴⁺-OH₂ bond or a long Mo⁴⁺-OH bond with mechanistic implications for substrate reduction.