Resonance Raman Spectroscopy of Pyranopterin Molybdenum Enzymes

Abstract

Resonance Raman spectroscopy (rR) is a powerful spectroscopic probe that is widely used for studying the geometric and electronic structure of metalloproteins. In this focused review, we detail how resonance Raman spectroscopy has contributed to a greater understanding of electronic structure, geometric structure, and the reaction mechanisms of pyranopterin molybdenum enzymes. The review focuses on the enzymes sulfite oxidase (SO), dimethyl sulfoxide reductase (DMSOR), xanthine oxidase (XO), and carbon monoxide dehydrogenase. Specifically, we highlight how Mo-O_oxo, Mo-S_sulfido, Mo-S_dithiolene, and dithiolene C=C vibrational modes, isotope and heavy atom perturbations, resonance enhancement, and associated Raman studies of small molecule analogs have provided detailed insight into the nature of these metalloenzyme active sites.

1. Introduction and Scope

This contribution focuses on how resonance Raman spectroscopy has contributed to our current understanding of pyranopterin enzyme geometric structure, electronic structure, and mechanism over the past 30 years. The review begins with a brief background of the canonical pyranopterin Mo enzymes sulfite oxidase (SO), xanthine oxidase (XO), and dimethyl sulfoxide reductase (DMSOR), in addition to the pyranopterin dithiolene ligand, which is the organic component of the Mo cofactor (Moco) and common to all these enzymes. Where appropriate, examples from resonance Raman studies of small model analogs have been included to enhance our understanding of the enzyme spectroscopic data.

2. Mo Enzyme Background

Pyranopterin Mo enzymes catalyze a wide variety of redox transformations that are based on oxygen atom transfer, hydroxyl/oxyl radical transfer, hydride transfer, and hydroxylation reactions involving their substrate molecules.^1–8 All these enzymes possess a unique molybdenum cofactor, or Moco, that is comprised of a Mo ion coordinated by the ene-1,2-ditholate linkage of the pyranopterin dithiolene (PDT, or MPT molybdopterin) ligand (Figure 1).⁹ A pyran ring connects the dithiolene chelate of the PDT with a pterin ring to yield an electronically complex ligand that has the potential to display a remarkable electronic flexibility.^10–15 Although a complete understanding of how the PDT functions in catalysis is still unknown, there is compelling evidence that the PDT functions to anchor Moco in the active sites of the enzymes, facilitate electron transfer reactivity, and modulate the reduction potential of the Mo site during the course of catalysis.¹⁰

Figure 1.

Structure of Moco derived from X-ray crystallographic and XAS/EXAFS studies of the Moco insertase. Moco is shown coordinated by a single pyranopterin dithiolene with a closed pyran ring.

Pyranopterin Mo enzymes are categorized as belonging to either the sulfite oxidase (SO) family, the xanthine oxidase (XO) family, or the dimethylsulfoxide reductase (DMSOR) family based on the nature of their protein folds, active site coordination geometry, or the nature of the reactions they catalyze.^{3, 7, 16} The relationship between these enzyme families and the PDT is underscored by the observation that out-of-plane PDT distortions appear to be related to enzyme function.¹⁷ Determining how the geometric structure of the PDT correlates with the Mo oxidation state,^{18, 19} and how the PDT contributes to the unique active site electronic structures of the enzyme active sites remains a very active area of research.

SO family enzymes.

The active site coordination geometry of SO family enzymes has been probed by EXAFS^{20, 21} and X-ray crystallography^22–27 to reveal a square pyramidal [(PDT)MoO₂(SR_Cys)]¹- geometry for SO_ox where the Mo(VI) ion is coordinated by a single PDT dithiolene, two terminal oxo ligands oriented cis to one another, and a S donor from a cysteine residue (Figure 2). The structure of SOred also possesses a square pyramidal geometry. In the oxygen atom transfer mechanism (Figure 3), it is the water-derived equatorial oxo (O_eq) ligand that is transferred to the substrate. Oxygen atom transfer is believed to occur via an associative mechanism, with the apical oxo (O_ap) functioning as a “spectator oxo”²⁸ in catalysis.^{1, 16, 29–35} The Mo=O_eq bond is oriented toward the substrate access channel, and the two-electron oxidation of the sulfite substrate is initiated by the attack of a sulfite S lone pair on O_eq to yield an initial E-P complex with sulfate bound to the reduced Mo(IV) center. This Mo-bound sulfate intermediate subsequently breaks down by product dissociation from the Mo ion and is followed by the binding of a water molecule to the reduced Mo(IV) site. The Mo(VI) form of the enzyme is regenerated from the reduced [(PDT)MoO(SR_Cys)(OH₂)]¹- Mo(IV) site by two sequential 1e⁻/1H⁺ transfers.

Figure 2.

The active site structure for Arabidopsis thaliana plant sulfite oxidase. PDB 1ogp.

Figure 3.

The generally accepted oxygen atom transfer mechanism proposed for sulfite oxidase.

DMSOR family enzymes.

Pyranopterin Mo enzymes that belong to the DMSO reductase family are commonly divided into three classes (i.e. Type I, II, and III) based on the nature of the non-PDT ligands bound the Mo ion.^{6, 7, 16} Unlike the SO family enzymes, which possess a single PDT ligand bound to Mo, the DMSO reductase family enzymes possess two PDT ligands that remain coordinated to the metal throughout the course of catalysis. The enzymes that comprise the DMSO reductase family are very diverse in nature and catalyze a wide range of chemical transformations. DMSO reductase family enzymes that lack additional redox chromophores are found among the Type III DMSO reductases, making them highly amenable to optical spectroscopies that can intimately probe their electronic structures.^{36, 37} As such, resonance Raman studies have focused on the Type III enzymes from Rhodobacter. Type III DMSO reductases have been structurally characterized by a combination of EXAFS^38–42 and X-ray crystallography^43–46 and display a [(PDT)₂MoO(O_Ser)]¹-distorted trigonal prismatic active site geometry in the oxidized state with two coordinated PDTs, a terminal oxo, and a coordinated serinate oxygen (Figure 4). Reduced forms of this active site have been structurally characterized by EXAFS and display Mo(IV) des-oxo [(PDT)₂Mo(OH₂)(O_Ser)]¹- and Mo(V) [(PDT)₂Mo(OH)(O_Ser)]¹- active site coordination spheres.⁴⁷ The source of the terminal oxo ligand derives from the substrate in an oxygen atom transfer reaction (Figure 5) that has been addressed both experimentally and computationally.^{31, 36, 42, 48–53} Here, catalysis is initiated in the reduced Mo(IV) state, with the substrate (DMSO) binding to Mo and replacing the labile water ligand. Oxygen atom transfer to Mo results in the two-electron oxidation of the metal to form product bound [(PDT)₂Mo(O-DMS)(O_Ser)]¹-, which is followed by product release that generates an oxidized [(PDT)₂MoO(O_Ser)]¹- site. The catalytically competent reduced form of the enzyme is generated by coupled e−/H⁺ transfer steps. The first of these converts [(PDT)₂MoO(O_Ser)]¹- to Mo(V) [(PDT)₂Mo(OH)(O_Ser)]¹-, which is also known as the high-g split intermediate due to its unique hydroxyl proton hyperfine interaction with ^95,97Mo.^{36, 37, 42, 54} A second e−/H⁺ transfer step converts high-g split to the fully reduced des-oxo [(PDT)₂Mo(OH₂)(O_Ser)]¹- state, with the loss of water generating five-coordinate [(PDT)₂Mo(O_Ser)]¹- as a putative resting state. A six-coordinate des-oxo [(PDT)₂Mo(OSMe₂)(O_Ser)]¹- results upon the binding of substrate, leading to the next catalytic cycle.³⁶

Figure 4.

The active site structures for oxidized (DMSOR_ox) and reduced (DMSOR_red) Rhodobacter sphaeroides DMSO reductase, each showing two PDTs coordinated to the Mo ion. Top: PDB 1eu1, Bottom: PDB 1h5n.

Figure 5.

The generally accepted oxygen atom transfer mechanism proposed for dimethylsulfoxide reductase.

XO family enzymes

The XO family enzymes xanthine oxidase/dehydrogenase (XO/XDH) and aldehyde oxidase (AO) have been structurally characterized by EXAFS and X-ray crystallography (Figure 6), and they are found to adopt 5-coordinate square pyramidal coordination geometries in both their oxidized and reduced forms.^55–63 The oxidized forms of these enzymes possess a catalytically essential terminal sulfido ligand, which is converted to a sulfhydryl donor following substrate oxidation. Importantly, metal activated water present as a hydroxide donor is bound to the oxidized Mo(VI) form of these enzymes. This coordinated hydroxide ligand is directed toward the substrate access channel and is in close proximity to a catalytically essential glutamate residue, which serves as an active site base to promote Mo-OH base-assisted nucleophilic attack on a carbon atom of a wide variety of substrates that include purines, various N-heterocycles, and aldehydes. The mechanism of the oxidation reaction catalyzed by XO family enzymes is markedly different than that of typical Fe and Cu monooxygenases.^{64, 65} Namely, the mechanism is not radical-based, the enzymes generate rather than consume redox equivalents, and water rather than dioxygen is the source of the oxygen atom incorporated into product. The nature of the cis-[MoOS]²⁺ unit and its function in catalysis has been studied both spectroscopically^{66, 67} and by various computational approaches,^{4, 29, 68–73} and this has contributed greatly to our understanding of the reaction mechanism of these enzymes. It is found that catalysis begins with base-assisted nucleophilic attack of the coordinated hydroxyl ligand on a substrate carbon center resulting in the formation of a tetrahedral intermediate and transition state that breaks down by formal hydride transfer to the terminal sulfido (Figure 7). For heterocyclic substrates, this yields a Mo(IV) E-P complex with the product bound to Mo as the enolate tautomer prior to product release. In the electron transfer half reaction, two sequential 1e⁻/1H⁺ transfers regenerate the oxidized [(PDT)MoOS(OH)]^1- form of the enzyme.

Figure 6.

The active site structure for bovine milk xanthine dehydrogenase with bound FYX-051 inhibitor. PDB 1v97.

Figure 7.

The generally accepted hydroxylation mechanism proposed for xanthine oxidase/dehydrogenase.

Another member of the XO enzyme family is the Mo-dependent carbon monoxide dehydrogenase,^{2, 5, 74–79} which converts CO to CO₂. Similar to XO/XDH and AO, this enzyme also adopts a square pyramidal coordination geometry with a coordinated PDT dithiolene, apical and equatorial oxo ligands, and a µ-sulfido ligand that bridges the Mo ion to a Cu(I) center that is also ligated to a cysteine thiolate (Figure 8).^{78, 79} Additionally, a water molecule may be weakly bound to the Cu site. Mechanistically, the Mo-dependent CODH catalyzes the oxidation of CO in a manner that is different than that of the Ni-Fe CODHs.^{4, 5, 74, 76, 78, 80–82} One of the proposed mechanisms for Mo-CODH is based on a detailed DFT and natural bond orbital (NBO) study, and begins with CO binding to Cu(I), followed by nucleophilic attack of the Mo-oxo oxygen on the carbonyl carbon to yield the cyclic intermediate shown in Figure 9.^{4, 5} Metal activated water coordinated to the Cu ion is poised for nucleophilic attack on the µ₂-η² CO₂ carbon center to yield bicarbonate bound to Mo(IV), which subsequently breaks down to release CO₂ as the ultimate product of the reaction. EPR spectroscopy using ¹³C labeled bicarbonate indicates that bicarbonate is not likely to be coordinated to the Mo(V) form of the enzyme.⁷⁵ Alternative mechanisms have been suggested that involve either the direct release of CO₂,^{7, 75, 76, 80} or the formation of a C-S bond along the reaction coordinate.^{74, 78, 82} The latter mechanism is based upon the crystal structure of an inhibited enzyme species.⁷⁹

Figure 8.

The active site structure for carbon monoxide dehydrogenase. PDB 1n63.

Figure 9.

Top: A mechanism proposed for carbon monoxide dehydrogenase that does not include an intermediate with a C-S bond. Bottom: A proposed µ- thiocarbonate intermediate suggested from the X-ray structure of an inhibited enzyme species.

3. Resonance Raman Spectroscopy

Background.

Resonance Raman spectroscopy is an important probe of metalloenzyme geometric and electronic structure,⁸³ and has played pivotal roles in increasing our understanding of pyranopterin containing molybdenum enzymes.^{1, 16, 84} In infrared spectroscopy, the selection rules are such that transitions are observed when there is a change in the dipole moment of a molecule as a function of the normal mode of vibration. Raman spectroscopy is complementary to infrared in that they both are used to probe molecular vibrations, but the Raman selection rules are such that vibrational transitions are observed when there is a change in the polarizability of the molecule as function of the normal mode of vibration. Key advantages of Raman over infrared spectroscopy for metalloproteins include its ease of use for aqueous samples, since water is a weak Raman scatterer, and the observed signal enhancement due to the resonance Raman effect. However, the interpretation of resonance Raman data can be complicated when the sample is heterogeneous, there are other strongly absorbing chromophores present in the sample, or the sample suffers from background fluorescence. Raman intensity enhancement derives from Franck-Condon scattering and Herzberg-Teller vibronic coupling.^{83, 85} In the case of Franck-Condon scattering (Albrecht A-term⁸⁵) Raman enhancement derives from the displacement of an excited state potential energy surface relative to that of the ground state along a normal coordinate of vibration, Q_k. Herzberg-Teller (Albrecht B-term⁸⁵) Raman enhancement occurs when the resonant excited state can mix with another excited state by vibronic coupling. Typically, the Franck-Condon A-term is the dominant contributor to the scattering under resonant conditions, leading to a large resonance enhancement of totally symmetric normal modes that collectively describe the nature of the excited state distortion coordinate. Detailed analyses of resonance Raman spectra have traditionally utilized a Kramers-Heisenberg sum over states formalism,⁸⁵ but time-dependent wavepacket approaches⁸⁶ have increased in popularity and have recently been used to understand resonance Raman enhancement in xanthine oxidase.⁸⁷

Model studies of Mo-S and Mo-oxo vibrations.

Perhaps the most characterized benchmark oxomolybdenum dithiolene complexes are Tp*MoO(bdt),⁸⁸ Tp*MoO(tdt),⁸⁸ and Tp*MoO(qdt)⁸⁹ (Tp* = hydrotris-(3,5-dimethyl-1-pyrazolyl)borate; bdt = 1,2-benzenedithiolate; tdt = 3,4-toluenedithiolate; qdt = quinoxaline dithiolate). These paramagnetic d¹ Mo(V) complexes are important for understanding the electronic structure of molybdoenzymes that possess a single terminal Mo≡O bond oriented cis to a dithiolene chelate, since this coordination geometry is encountered in many pyranopterin containing Mo enzyme forms.^{1–3, 6, 16, 19, 88, 90} The [MoO(dithiolene)]⁻¹ unit in these molecules possesses idealized C_s symmetry, with the mirror plane bisecting the two S atoms of the dithiolene and containing the Mo≡O unit. From a resonance Raman spectroscopy perspective, the presence of a symmetry element simplifies the analysis of the spectra since there are a’ (symmetric) and a” (asymmetric) vibrational modes. Within this 4-atom core, there are 6 vibrational modes. These include the symmetric Mo≡O (ν_Mo≡O) and S-Mo-S (ν_Mo-S) stretching vibrations, the symmetric bend (δ_S-Mo-S), the asymmetric stretch (ν_Mo-S), the symmetric wag (ω), and the asymmetric twist (τ). A combination of qualitative rR depolarization ratios⁸⁸ and rR excitation profiles for Tp*MoO(bdt) showed that excitation into dithiolene → Mo(xy) LMCT transitions, which directly probe the redox orbital, resulted in resonance enhancement of two totally symmetric vibrations, the symmetric δ_S-Mo-S bend at 362 cm⁻¹ and the ν_Mo-S symmetric stretch at 393 cm⁻¹ (S-Mo-S stretch), while excitation into dithiolene → Mo(xz,yz) LMCT transitions resulted in resonance enhancement of the symmetric ν_Mo≡O stretch at 932 cm⁻¹ (Figure 10). The resonance Raman results for Tp*MoO(qdt) were similar to those of Tp*MoO(bdt), displaying vibrational bands at 348 and 407 cm⁻¹. However, a computational analysis of the normal modes showed that the S-Mo-S (ν_Mo-S) stretch and bend (δ_S-Mo-S) vibrations were mixed, leading to a different normal mode description for the MoOS2 core.⁸⁹ A key observation from these model studies is the small number of vibrational modes in the low-frequency (~200–1000 cm⁻¹) region of the Raman spectra, which is in stark contrast to what is observed in many of the enzymes, where low-frequency Mo-S vibrations can be coupled with other low-frequency modes of the pyranopterin or even kinematically coupled with other protein modes. Considering bis-dithiolene complexes, the symmetrized Mo(QAd)(dithiolene)₂ (Ad = 2-adamantyl; Q = O, S, Se) displayed only a single vibration at ~400 cm⁻¹ in the 250 – 600 cm⁻¹ region of the Raman spectrum that was assigned as the totally symmetric MoS₄ (ν_Mo-S) stretching vibration.⁹¹ The Raman spectra of the related MoO(SPh)₄ and MoO(SPh-PhS)₂ complexes were more complex, displaying five vibrations below 600 cm⁻¹ in addition to the ν_Mo≡O stretch. Mo-S modes were tentatively assigned at 366 cm⁻¹ and 425 cm⁻¹ for MoO(SPh)₄ and 375 cm⁻¹ and 423 cm⁻¹ for MoO(SPh-PhS)₂.⁹²

Figure 10.

Resonance Raman spectrum of Tp*MoO(bdt) and associated symmetry coordinates. Adapted from ref. 80.

4. Resonance Raman Spectroscopy of Sulfite Oxidase Family Enzymes

Resonance Raman spectra were initially collected on both the wt and C207S trioxo variant forms of the human SO Mo domain in the late 1990s.^{93, 94} To obtain Mo domain specific Raman spectra without the strong interference that originates from the highly absorbing cytochrome b₅-type heme, the heme domain was removed by tryptic cleavage of the K108R and K108R/C207S mutants.⁹⁵ This seminal study provided the first Mo-oxo stretching vibrations for a pyranopterin Mo enzyme. Specifically, two Mo-oxo stretching vibrations were observed in SO_ox at 903 cm⁻¹ and 881 cm⁻¹ that shifted to 890 cm⁻¹ and 848 cm⁻¹ with redox cycling in H₂₁₈O buffer. The assignment of these bands as the symmetric ν_s (903 cm⁻¹) and asymmetric ν_as (881 cm⁻¹) O-Mo-O stretches was supported by prior studies on cis-dioxomolybdenum model compounds.^96–100 Based on a reduced mass argument, the observed isotopic shift was approximately half of that anticipated if both of the oxo groups were labeled by ¹⁸O, and this supports the conclusion that only one of the oxo groups is labile and capable of being labelled under these conditions. These two Mo-oxo vibrational modes were observed to be resonantly enhanced with excitation into the 480 nm charge transfer band that has been assigned as a S_Cys → Mo(VI) LMCT due to the absence of this absorption feature in the C207S variant.⁹⁵ Additionally, low-frequency rR bands were observed at 289 cm⁻¹ and 362 cm⁻¹ with excitation into this LMCT band, and they were assigned as the coordinated cysteine S_γ-C_β-C_α bending vibration and a Mo-S stretching vibration, respectively. High-frequency vibrations in resonance with the LMCT band were assigned to modes associated with the dithiolene chelate. These band were observed at 1006 cm⁻¹ and 1161 cm⁻¹ (coupled C-S and C-C stretches), and at 1532 cm⁻¹ (C=C stretch).

Later, resonance Raman studies were performed on sulfite oxidase as part of a study aimed at understanding electronic structure contributions to equatorial Mo-oxo bond activation in sulfite oxidizing enzymes.^{35, 101} In addition to the previously assigned 20,833 cm⁻¹ S_Cys → Mo LMCT band,^{93, 94, 101} the electronic absorption spectrum of oxidized wt plant sulfite oxidase (A. thaliana pSO) displays a higher energy band at 27,778 cm⁻¹ that can be assigned as a S_dithiolene → Mo LMCT transition.¹⁰¹ The one-electron promotions associated with these LMCT bands originate from filled ligand-based orbitals and are promoted to the LUMO. Early DFT computations on a small [Mo(VI)O₂(S₂C₂Me₂)(SCH₃)]- computational model for the SO_0x active site showed the dramatic inequivalence of the apical (O_ap) and equatorial (O_eq) oxo ligands due to a strong trans influence on O_eq.³⁵ This observation is in stark contrast to more symmetric cis-dioxo MoO₂ model complexes where the two oxo ligands are effectively related by a symmetry element.^96–100 This work also emphasized the critical importance of the SO_ox LUMO, since it possesses dominant Mo(xy)-O_eq π* antibonding character (52% Mo, 20% O_eq) with effectively no O_ap contribution.³⁵ The importance of the LUMO in the reductive half reaction of sulfite oxidizing enzymes stems from the fact that it is the two-electron acceptor orbital in oxygen atom transfer reactions with oxo acceptor substrates. Thus, the unique electronic structure of the site lowers the activation energy for Mo-O_eq bond cleavage along the reaction coordinate and promotes product release.³⁵

Due to the Mo(xy)-O_eq π* character of the SO LUMO, LMCT excitations that result in population of the LUMO are expected to produce an excited state distortion (i.e. bond lengthening) along the Mo-O_eq bond. Raman spectroscopic studies on A. thaliana plant SO (pSO) yielded strong experimental support for this hypothesis,¹⁰¹ confirming the critically important Mo(xy)-O_eq π* antibonding interaction in the LUMO of SO_0x suggested by the earlier computational study.³⁵ Low temperature (30K) Raman spectra collected on resonance (488 nm) with the S_Cys → Mo LMCT band displayed prominent vibrational bands at 896, 877, and 864 cm⁻¹ (Figure 11). A combination of DFT frequency computations on both large and small molecule active site models, combined with an ¹⁸O isotope perturbation, allowed for the higher frequency peak at 896 cm⁻¹ to be assigned at the symmetric O-Mo-O stretch (ν_s) and the 864 cm⁻¹ band to be assigned as the antisymmetric O-Mo-O stretch (ν_as). Both of these stretches possess noticeable mode localization due the low-symmetry of the oxidized active site. In a small computational model of the active site, the 877 cm⁻¹ vibration was shown to possesses a dominant combination of Mo-O_eq and symmetric C-S dithiolene stretching character. In contrast, for cis-MoO₂ complexes with symmetry related oxo donors, one observes strong resonance enhancement of the totally symmetric O-Mo-O stretch, but the antisymmetric O_oxo-Mo-O_oxo stretch possesses markedly reduced Raman intensity. The rationale for resonance enhancement of both the ν_s and ν_as O_oxo-Mo-O_oxo stretches in SO_0x derives from the unique nature of the active site LUMO. As mentioned previously, one-electron promotions to the LUMO result in an excited state distortion only along the Mo-O_eq bond, which can be described as a linear combination of the ν_s and ν_as Mo-oxo stretches, and the observation of resonance enhancement for both modes. By analogy, the 864 cm⁻¹ mode is also observed to be resonantly enhanced since there is Mo-O_eq stretching character in this vibration. Thus, the rR results are consistent with the 20,833 cm⁻¹ band in SO_ox being assigned as a S → Mo(xy)-O_eq π* LMCT transition, contributing to a greater understanding of the SO_ox LUMO and Mo-O_eq bond activation along the reaction coordinate when this orbital becomes doubly occupied in the reductive half reaction.¹⁰¹

Figure 11.

Resonance Raman spectrum of as-prepared A. thaliana plant SO. The spectrum is virtually identical the that of oxidized A. thaliana pSO redox cycled in H₂¹⁸O. Adapted from ref. 94.

Regarding electron transfer reactivity in the oxidative half reaction of SO, a combined cyclic voltammetry and surface enhanced resonance Raman (SERR) study of human sulfite oxidase (hSO) was used to probe heterogeneous electron transfer and catalysis in this enzyme, and it was shown that this activity was a function of the local protein environment.¹⁰² Specifically, electron transfer from the reduced Mo site to the electrode requires the Cyt b5 domain to change its orientation from one where it is interacting with the Mo domain to a new orientation where it can interact with the self-assembled monolayer (SAM) surface. The Mo and Cyt b5 domains are connected via a flexible polypeptide loop, which facilitates intraprotein electron transfer between these redox centers when the heme domain interacts with the surface of the Mo domain.¹⁰³ Interprotein electron transfer is facilitated by docking of the heme domain with its electron transfer partner Cyt c. In this study, where the heme domain interacts with the electrode, a very fast heterogeneous electron transfer rate (ks = 440 s⁻¹) was measured that displayed a dependence on ionic strength. Fast electron transfer was determined to be a result of a shorter contact time with the SAM surface at higher ionic strength.¹⁰²

5. Resonance Raman Spectroscopy of DMSO Reductase Family Enzymes

DMSO reductase family enzymes can be divided into three basic types based on their structure and the nature of the non-PDT ligands that are bound to the Mo ion.^{1, 6, 7, 16} However, only the type III DMSOR family enzymes, which possess a general [MoO(PDT)₂(OR)]^1- (OR = serinate, aspartate) oxidized enzyme active site structure, have been probed in detail by resonance Raman spectroscopy. These Type III enzymes generally lack the strongly absorbing flavin, heme, and Fe-S cluster redox chromophores that are present in many other enzymes. These redox chromophores obscure the weaker electronic absorption features of the Mo active site and effectively render resonance Raman interrogation of the active site difficult or impossible. R. sphaeroides DMSOR is a typical type III enzyme and possesses distinct low energy charge transfer features at 720 nm for DMSOR_ox and 640 nm for DMSOR_red.^{37, 93, 94} These isolated absorption bands conveniently allow for Raman spectra to be collected on-resonance with these low-energy transitions using the 676.4 nm line of a Kr+ laser. Initial studies by Spiro and coworkers¹⁰⁴ showed that excitation into these low energy charge transfer bands of DMSOR_ox produced rR enhancement of vibrations in the PDT dithiolene C=C stretching region centered at 1575 cm⁻¹. They showed that the C=C stretching frequency is downshifted by ~ 7 cm⁻¹ to 1568 cm⁻¹ for DMSOR_red, consistent with a change in Mo-dithiolene covalency as a function of the Mo ion oxidation state. The dithiolene C=C stretching vibration is expected to be observed in the 1300 – 1600 cm⁻¹ region of the Raman spectrum^{100, 105} with frequencies that are anticipated to vary as a function of the oxidation state of the dithiolene,^{12, 13, 106, 107} the oxidation state of the metal, and the magnitude of the “sulfur-fold” distortion within the five-membered chelate ring.^{18, 89, 106} In 1995 Spiro and coworkers noted that the 1568 cm⁻¹ band that had previously been observed for DMSOR_red, and assigned as a C=C stretching vibration, was likely an experimental artifact.¹⁰⁸ This latter study assigned the 1576 cm⁻¹ mode observed for DMSOR_ox as a C=C stretch from a 5,8-dihydro form of the pterin component of the PDT, with a 1526 cm⁻¹ band being assigned as the PDT dithiolene C=C stretch.

R. sphaeroides DMSOR isolated from bacteria grown on 34S was used to understand the nature of low-frequency vibrational modes associated with the dithiolene component of the PDT.¹⁰⁴ Two ³⁴S sensitive bands were observed for DMSOR_ox at 350 cm⁻¹ and 370 cm⁻¹, which shift to 352 cm⁻¹ and 383 cm⁻¹ in DMSOR_red. These bands were assigned as arising from the symmetric and asymmetric S-Mo-S stretches of the dithiolene chelate. Additional low-frequency Raman bands were also observed to be affected by the 34S isotope perturbation. This work was important, since the observation of both C=C and Mo-S stretching vibrations in resonance with a low-energy CT transition were used to confirm that the dithiolene component of the PDT was directly coordinated in a bidentate fashion to the Mo ion. ³⁴S labeling of the enzyme indicated that, in addition to Mo-S stretches, other vibrational modes in the 250 – 420 cm⁻¹ region are present that couple with the Mo-S stretching vibrations leading to their resonance enhancement.¹⁰⁸ Resonance Raman studies of both pterin and quinoxoline containing model compounds show isotope sensitive bands that shift by 7–8 cm⁻¹, supporting the idea of extensive low-frequency vibrational mode mixing in DMSOR.¹⁰⁸ Terminal Mo-oxo stretches are typically observed between 850 – 1000 cm⁻¹.^{88, 108} Although weak rR bands had been observed in this spectral region for R. sphaeroides DMSOR, these early studies indicated that redox cycling in the presence of ¹⁸OH₂ did not result any frequency shifts associated with the isotopic labeling.¹⁰⁸

Later, a more complete resonance Raman study of four R. spaeroides DMSOR enzyme forms was performed that allowed for all of the vibrational modes observed in the 200 – 1700 cm⁻¹ region to be assigned as originating from Mo≡O, dithiolene, Mo-O_Ser, Mo-S, and DMSO vibrational modes or non-resonantly enhanced modes of the protein (Figure 12).^{93, 109} This study, conducted after the initial crystal structure was published for DMSO reductase,⁴⁶ probed DMSOR_ox, DMSOR_red, DMS reduced DMSORred, and a glycerol inhibited Mo(V) form that is structurally similar to the high-g split paramagnetic intermediate that has been studied extensively by MCD, electronic absorption, and EPR spectroscopies,³⁶ in addition to EXAFS.^{42, 54} An ¹⁸O isotopic perturbation study on the Mo(VI) form of the enzyme was used to assign the ν_Mo≡O stretch at 862 cm⁻¹ and show that DMSOR0x is a mono-oxo species, consistent with oxygen atom transfer from the substrate to a des-oxo Mo(IV) species. The vibrational frequencies and vibrational mode assignments for the four different enzyme forms have been summarized.¹⁰⁹ DMS reduced DMSOR was shown to possess a bound DMSO product molecule, as evidenced by ν_Mo-O and ν_S=O stretches observed at 497 cm⁻¹ and 862 cm⁻¹ respectively, again consistent with an associative oxygen atom transfer mechanism.^{36, 109} The X-ray structures of DMSORs at that time yielded markedly different descriptions of the first coordination sphere ligation to the Mo ion. The structure of the R. sphaeroides enzyme⁴⁶ indicated a mono-oxo Mo(VI) species with one of the PDT dithiolenes being asymmetrically coordinated to the metal with a long Mo-S_dithiolene bond length of 3.1Å. The reduced des-oxo R. sphaeroides structure revealed partial dissociation of one of the PDT dithiolene ligands. The DMSOR_ox structure from R. capsulatus indicated a di-oxo Mo(VI) site with one of the dithiolene ligands being completely de-coordinated from Mo.⁴⁴ Thus, the resonance Raman work¹⁰⁹ is of considerable importance since it definitively showed that all four of the Mo-S_dithiolene bonds remained coordinated in the different enzyme forms studied, the Mo(VI) enzyme form was a mono-oxo species, and the Mo(IV) form was a des-oxo species clearly supporting an oxygen atom transfer mechanism.

Figure 12.

Resonance Raman spectrum of R. sphaeroides DMSOR. Adapted with permission from Garton, S. D.; Hilton, J.; Oku, H.; Crouse, B. R.; Rajagopalan, K. V.; Johnson, M. K., Active Site Structures and Catalytic Mechanism of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase as Revealed by Resonance Raman Spectroscopy. J. Am. Chem. Soc. 1997, 119 (52), 12906–12916.

Biotin sulfoxide reductase (BSOR) is another DMSO reductase family enzyme that is closely related to the R. capsulatus and R. sphaeroides DMSORs. Resonance Raman data for BSOR have been collected for as-prepared DMSOR_ox, product-associated DMSOR_ox, and dithionite-reduced DMSOR_red.¹¹⁰ The Mo-S stretching vibrations (200 – 600 cm⁻¹) for R. sphaeroides biotin sulfoxide reductase have been analyzed in the context of an idealized square pyramidal C_4v site. Resonance Raman spectra for as-prepared BSOR_ox, using excitation wavelengths between 457–647 nm allowed for assignment of the totally symmetric Mo-S₄ breathing mode at 355 cm⁻¹. This is the dominant spectral feature in the Mo-S stretching region and can be compared with the corresponding vibrational mode in DMSOR_ox at 350 cm⁻¹. Similarly, for reduced BSOR_red and DMSOR_red these modes were assigned at 363 cm⁻¹ and 367 cm⁻¹, respectively. The observation of strong resonance enhancement for these breathing modes with laser excitation across the visible and NIR spectral region was used to suggest the optical bands in this region could be assigned as dithiolene→Mo(VI) LMCT transitions, in agreement with spectral assignments for DMSOR.^{93, 109}

A comparison of the observed and assigned Mo-S stretching vibrations for DMSOR and BSOR clearly show that active sites of these prokaryotic oxotransferase enzymes are indeed very similar. Additional vibrational bands at 288, 416, and 447 cm⁻¹ were not observed to shift significantly as a function of oxidation state or dithiolene ³⁴S isotopic substitution and have therefore been assigned as originating from deformations within the PDT dithiolene and pyran rings. Similar assignments were previously made for DMSOR.¹⁰⁹ Raman bands at 463 cm⁻¹ and 543 cm⁻¹ were suggested as potential Mo-O_Ser stretching modes in an as-prepared form of the enzyme (i.e. BSOR_ox), and Raman bands at 425 cm⁻¹ and 495 cm⁻¹ were suggested for the Mo-O_Ser stretch in BSOR_red. For comparison, the Mo-O_Ser stretching vibration was tentatively assigned at 513 cm⁻¹ for DMSOR_red and at 536 cm⁻¹ for DMSOR_ox.¹⁰⁹ Serine coordination is of current interest in DMSOR and related Type III enzymes since this ligand has been suggested to be labile. Current evidence for the coordination of serine in the Mo(V) state derives from a combined electronic absorption, MCD, EPR, and computational study of the high-g split catalytic intermediate.⁴² Obtaining direct evidence from EXAFS for a Mo(V)-O_Ser scatterer is more problematic, but the recent model studies suggest that serine does remain coordinated throughout the catalytic cycle.⁴²

The Mo≡O stretch for BSOR0x is observed at 860 cm⁻¹ and this band shifts to 818 cm⁻¹ after redox cycling in H₂¹⁸O buffer, as predicted using the reduced mass for a diatomic Mo≡O oscillator.¹¹⁰ This is consistent with a mono-oxo Mo(VI) site in BSOR0x. As expected, the mono-oxo Mo≡O stretching vibration is not observed in reduced enzyme, in full accord with a des-oxo Mo(IV) site. Dithionite reduced BSOR that was subsequently oxidized using ¹⁶O DMSO results in a mono-oxo Mo site that possesses peaks at 840 cm⁻¹ and 860 cm⁻¹ that were assigned as Mo≡O stretching vibrations, with the former being more intense than the latter using 568 nm excitation. The observation of two Mo≡O stretching vibrations was used to support an argument for active site heterogeneity that results from interactions between the protein (e.g. Trp-90 in BSOR) and the terminal oxo ligand, indicating a degree of active site conformational flexibility.¹¹⁰ Although redox cycling did not seem to affect this active site heterogeneity, the observation of the Mo≡O stretching band sharpening during catalytic cycling showed that this heterogeneity could be reduced to yield an active site with less conformational flexibility. Due to the difficulty in synthesizing ¹⁸O labeled BSO, Me₂S¹⁸O was used to label BSOR under turnover conditions. An elegant set of ¹⁶O/¹⁸O labeling experiments showed that during catalytic cycling with the non-physiological Me₂SO substrate, the terminal oxo ligand in the Mo(VI) state can exchange with both water and the oxygen atom of Me₂SO. Furthermore, analysis of the BSOR Mo≡O stretch allowed for a description of how the enzyme interacts with various product molecules that appear to be weakly bound to the Mo(VI) ion in the active site (Figure 13).

Figure 13.

R. sphaeroides BSOR structures derived from resonance Raman spectroscopy. Adapted from ref. 106.

Raman spectroscopy is a very sensitive probe of the nature of the dithiolene that is bound to the Mo ion in both the proteins and models. Namely, the ν(C-S) and ν(C=C) modes are found to be inversely correlated,^{84, 105, 110, 111} with high ν(C=C) and low ν(C-S) frequencies indicative of dithiolate character and low ν(C=C) and high ν(C-S) frequencies signifying a greater degree of dithione character in the chelate ring.¹¹⁰ For BSOR, vibrations in the ν(C=C) stretching region were used to propose that the enzyme possesses two different types of PDT dithiolene ligands, as was also noted for as-prepared wild-type DMSOR.¹⁰⁹ Similarly, the resonance Raman spectra of the DMSOR family enzyme arsenite oxidase was interpreted in the context of one PDT dithiolene possessing a dithiolate structure with the second having more π-delocalization.¹¹² Model studies of oxo-Mo(IV) dithiolene complexes display evidence of thiol/thione character in a single dithiolene,^{12, 13} complicating the issue of ν(C-S) and ν(C=C) vibrational assignments and highlighting aspects of the non-innocent nature of the dithiolene when bound to Mo.^{1, 10, 12–14, 19, 90, 107}

Raman data for Type III DMSOR family enzymes can be compared with the Raman spectra of good structural models (e.g. [Mo^VIO(OSiPr₃)(S₂C₂(COOMe)₂)₂]-) for the DMSOR0x site, and these studies¹¹³ suggest an alternative proposal to the original idea that DMSO reductase possesses one PDT dithiolene that has dithiolate character with the second being more π-delocalized.¹⁰⁹ The Mo^VIO(OSiPr₃)(S₂C₂(COOMe)₂)₂]- model possess terminal oxo ligation, a serine mimic (i.e. OSiPr3), and two coordinated true olefinic dithiolene chelates.¹¹⁴ Remarkably, these complexes also are good electronic structural models for the DMSOR_ox site since the electronic absorption spectrum of [Mo^VIO(OSiPr₃)(S₂C₂(COOMe)₂)₂]- possesses low energy charge transfer bands at 13 533 cm⁻¹ (ε=1350 M⁻¹ cm⁻¹) and 17 549 cm⁻¹ (ε=2800 M⁻¹ cm⁻¹), that are very similar in intensity and transition energy to those observed for DMSOR_ox.^{93, 109} The resonance Raman spectrum of [Mo^VIO(OSiPr₃)(S₂C₂(COOMe)₂)₂]^1- shows dithiolene C=C stretches at 1554 and 1489 cm⁻¹,¹¹⁴ which are slightly lower than the frequencies observed for R. sphaeroides DMSO reductase (1578 and 1527 cm–1).⁹³ Vibrational frequency computations performed on [Mo^VIO(OSiPr₃)(S₂C₂(COOMe)₂)₂]^1- show dithiolene C=C stretches at 1577 and 1505 cm⁻¹, which were assigned as arising from each dithiolene ligand (Figure 14). The two dithiolenes are not symmetry related, with one of the dithiolenes (A) oriented such that both S donors are in the equatorial plane of the complex, while the second dithiolene (B) is oriented such that only one S donor is in the equatorial plane with the second oriented trans to the apical oxo ligand. This geometry results in a unique Mo-S π*-bonding interaction^{114, 115} in the model system that leads to a LUMO that possesses ~18% S character from the (B) dithiolene. This same Mo-S π*-bonding interaction can be inferred to occur in DMSORs and BSOR due to the similarity of their electronic absorption and resonance Raman spectra with those of the model. The implication of this type of covalent bonding interaction in DMSO reductase suggests that the PDT with the lower frequency dithiolene C=C stretch likely possesses a dithiolene with a single S donor in the equatorial plane and an ~180° O_oxo-Mo-S_dithiolene-C dihedral angle. Thus, this dithiolene could function to provide a hole superexchange pathway for electron transfer regeneration of the reduced catalytically competent DMSOR_red site involving a π-type bonding scheme similar to that observed in blue copper proteins.^{116, 117} Although there is still much to be learned regarding the nature of the PDT in DMSOR family enzymes, the resonance Raman spectra for DMSOR, BSOR, and model systems support a high degree of Mo coordination sphere structural similarity between these two enzymes and a similar oxygen atom transfer reaction mechanism with the Mo ion cycling between the Mo(IV) and Mo(VI) oxidation states.

Figure 14.

Resonance Raman spectrum of the DMSOR/BSOR model compounds [Mo^VIO(OSiBuPh₂)(S₂C₂(COOMe)₂)₂]^1- (a) and [MoVIO(OSiPr₃)(S₂C₂(COOMe)₂)₂]^1- (b). Adapted with permission from Sugimoto, H.; Tatemoto, S.; Suyama, K.; Miyake, H.; Mtei, R. P.; Itoh, S.; Kirk, M. L., Monooxomolybdenum(VI) Complexes Possessing Olefinic Dithiolene Ligands: Probing Mo-S Covalency Contributions to Electron Transfer in Dimethyl Sulfoxide Reductase Family Molybdoenzymes. Inorg. Chem. 2010, 49 (12), 5368–5370.

6. Raman of Xanthine oxidase family enzymes

In the early 1980s, Palmer and coworkers,^{118, 119} used XO to catalyze the oxidation of lumazine to violapterin and form a stable charge transfer complex that possesses an intense long wavelength absorption feature at 650nm (15,385 cm⁻¹).¹²⁰ Initial resonance Raman experiments on XO were obtained by optically exciting into the long-wavelength absorption of the enzyme complexed with violopterin.¹²⁰ This work was important since resonance Raman studies on XO family enzymes are made difficult by the presence of FAD and 2Fe2S ferredoxin clusters. These redox chromophores conspire to mask the Mo active site absorbance due to their greater extinction coefficients and contributions to background fluorescence. The low energy absorption band in the charge transfer complex was originally assigned as a Mo→violapterin charge transfer transition, with violapterin being oriented cis to the Mo≡O bond. This assignment was based on the ν_Mo≡O stretch not being resonantly enhanced with excitation into this band. Although the ν_Mo≡O stretch was not resonantly enhanced, numerous violapterin vibrational modes were observed to be resonantly enhanced using 676.4 nm excitation. Additionally, vibrational bands between 250–1100 cm⁻¹ were also observed and hypothesized to originate from the Mo coordination sphere. A ^16,18O isotope perturbation was used to tentatively assign the 276, 517, and 853 cm⁻¹ bands as originating from the Mo-O-R unit of the coordinated violapterin. In subsequent work, Hille and coworkers again used 676.4 nm laser excitation to excite into this long wavelength charge transfer band and observed strong resonance enhancement of Raman vibrations deriving from violopterin product modes.¹²¹ A combination of ^16,18O and ^1,2H isotope perturbations, coupled with DFT frequency calculations, were used to make extensive vibrational frequency assignments in the 350–1750 cm⁻¹ region of the spectra. These data convincingly showed that the violopterin product was bound to Mo in an end-on geometry, with the oxygen atom of the Mo-O-R_violopterin bridging unit being introduced by direct hydroxylation of the lumazine substrate. Due to the large number of vibrational modes that possess a bridging oxygen contribution, a side-on binding mode of product was eliminated as a possibility.

This work was followed by additional resonance Raman spectroscopic studies by Kirk and coworkers^{87, 122–124} who employed the reducing substrates 4-thiolumazine and 2,4-dithiolumazine in place of lumazine. Enzymatic turnover of these thiolumazine substrates generated new Mo(IV)-product complexes (Mo(IV)-4TV and Mo(IV)-2,4TV) that possessed a single intense long wavelength absorption feature in the near-infrared region of the spectrum (758–778 nm) using both bovine xanthine oxidase (XO) and Rhodobacter capsulatus xanthine dehydrogenase (XDH). Using these heavy thiolumazine congeners of lumazine, the MLCT band was shifted to markedly lower energy relative to Mo(IV)-product complexes that used lumazine as the reducing substrate.^{121, 125} This allowed for the acquisition of high quality resonance Raman spectra using 780nm excitation that were completely devoid of contributions from the 2Fe2S and FAD chromophores, and the ability to separate low-frequency violapterin product-based vibrational modes from those of the enzyme active site. The 200–600 cm⁻¹ Raman spectra for XO and XDH were virtually identical and displayed a combination of Mo-PDT modes and in-plane bending modes of the bound product. Additional electronic absorption and resonance Raman studies were performed using 4-thiolumazine and 2,4-dithiolumazine on wt XDH from R. capsulatus and the Q197A and Q102G variants.¹²⁴ Q197A and Q102 potentially hydrogen bond to the terminal oxygen of the Mo≡O group and the amino terminus of the PDT, respectively. It was observed that both the energy and the band shape of the Mo(IV) → product charge transfer transition are virtually unchanged as a function of the Q197A and Q102G variants, providing evidence that these residues do not dramatically affect the bonding in these Mo(IV)-thiolumazine product complexes. Moco specific vibrational modes were observed for Mo(IV)-4TV at 234, 290, 326, and 351 cm⁻¹ (Bands A-D) that were also observed in Mo(IV)-2,4TV) at 236, 286, 326, and 351 cm⁻¹ (Figure 15). DFT frequency computations were used to assist in the frequency assignments, with Band A being assigned as arising from a dithiolene envelope fold with Mo≡O rocking and pyranopterin contributions, and Band B being assigned as an asymmetric dithiolene ring distortion with Mo-SH stretching and pyranopterin contributions. Band C possessed a dominant symmetric S-Mo-S dithiolene core stretching and bending component with some Mo≡O rocking and pyranopterin contributions, while Band D was described as having a combination of S-Mo-S dithiolene and Mo-SH stretching contributions. These low frequency Moco vibrations derive from the nature of the Mo(IV) → product excited state distortion, which is driven by the fact that this charge transfer excited state possesses a large degree of hole character on the Mo ion. The observation of these resonantly enhanced Moco modes provide evidence that the PDT serves as an electron transfer conduit in the oxidative half-reactions of these enzymes and that the PDT is quite sensitive to redox changes at the Mo center.^{88, 123, 124}

Figure 15.

Low frequency resonance Raman spectra of wt XDH from R. capsulatus and the Q197A and Q102G variants. Reproduced with permission from Dong, C.; Yang, J.; Reschke, S.; Leimkühler, S.; Kirk, M. L., Vibrational Probes of Molybdenum Cofactor–Protein Interactions in Xanthine Dehydrogenase. Inorg. Chem. 2017, 56 (12), 6830–6837.

A combination of bonding and spectroscopic calculations have been used to understand the electronic origin of resonantly enhanced, high-frequency, in-plane product stretching vibrations in bovine Mo(IV)-4TV and Mo(IV)-2,4TV enzyme-product complexes (Figure 16).⁸⁷ Here, the low-energy absorption band was assigned as a Mo(xy) → product π* MLCT transition involving a one-electron promotion to the thiolumazine LUMO. The intensity of this MLCT band, coupled with the observation of strong resonance enhancement for in-plane 4TV and 2,4TV modes, was used to show that the apical Mo≡O bond must be oriented in the same plane as the product molecules. This allows for the doubly occupied Mo(xy) redox orbital to be involved in a π-bonding interaction with the π-LUMO of the product molecule. The resonance Raman enhancement of these thiolumazine modes results from excited state distortions within the Mo–O–C_product linkage and are therefore of key mechanistic importance since they can be correlated with a covalent pathway for electron flow between Mo and substrate.⁸⁷ More specifically, the tetrahedral intermediate and transition state^{57, 64, 72, 73, 126} possesses a Mo-O_eq-C_product linkage, which provides a direct π-orbital pathway for the two-electron oxidation of the thiolumazine and other heterocyclic substrates. This work formed the basis for later spectroscopic and computational studies on XO that showed specific electron delocalizations in the cis-[MoOS]²⁺ unit and the Mo-O_eq-C_substrate linkage make substantial contributions to C-H bond activation and transition state stabilization along the reaction coordinate.¹²⁷

Figure 16.

High frequency resonance Raman spectrum of bovine XO Mo(IV)-4-TV. Adapted from ref. 79.

Resonance Raman studies on both sulfo and desulfo forms of bovine XOox were used to assign metal-ligand vibrations in the oxidized enzyme.¹²⁸ Raman data collected using 400 – 650 nm excitation allowed identification of the Mo(VI)=S stretch at 474 cm⁻¹ in sulfo XOox, which was downshifted by 12 cm⁻¹ in the ³⁴S sulfo form. By comparison, the Mo(VI)≡O stretch was observed as a weak band at 899 cm⁻¹ in sulfo XOox. Raman excitation profiles indicated that both the Mo(VI)=S and Mo(VI)≡O stretches were maximally resonantly enhanced at 400 nm, and this was used to suggest that the 425 nm absorption feature, obtained by spectral subtraction of the desulfo electronic absorption spectrum from the sulfo spectrum, was a S_sulfido→ Mo LMCT band. However, it was acknowledged that the 2Fe2S centers and FAD dominantly contribute to the electronic absorption spectrum in the 360 – 470 nm region, making this charge transfer assignment tentative. Using 568.2 nm excitation, low frequency vibrations observed at 334, 348, 365, and 408 cm⁻¹ were suggested as possible Mo-PDT vibrational modes or Fe-S vibrations arising from the 2Fe2S clusters. These Mo ≡ O_oxo and Mo=S vibrational assignments can be compared with the model compound Tp^iPrMo^VIOS(OPh),⁶⁷ which possesses an apical oxo ligand oriented cis to the terminal sulfido donor in the equatorial plane as observed in XO_0x. The solution Raman spectrum of Tp^iPrMo^VIOS(OPh) revealed the Mo ≡O_oxo stretch at 912 cm⁻¹ and the Mo=S stretch at 486 cm⁻¹. It is important to note that ν_MoS is markedly less than that observed for Tp*MoSCl₂ (525 cm⁻¹),¹²⁹ which possesses a formally triple bonded terminal sulfido ligand. Thus, there is a significant reduction of the Mo-S bond order in the cis-[MoOS] unit due to the presence of the strong field terminal oxo ligand.⁶⁷

Resonance Raman Spectroscopy of CODH.

Resonance Raman spectra of CODH_ox from Oligotropha carboxidovorans were collected using 514.5 nm excitation and displayed vibrational modes that can be associated with the Mo active site, FAD (1300–1400 cm⁻¹) and Fe/S clusters (300–350 cm⁻¹) (Figure 17).⁷⁶ Vibrational modes that may be assigned as originating from the [(PDT)MoO₂-SCu(S_Cys)]²- bimetallic site are observed at 895, 879, and 861 cm⁻¹, in remarkable agreement with the Mo-O_oxo stretching vibrations observed for A. thaliana sulfite oxidase.¹⁰¹ Using this spectral comparison between enzymes that possess di-oxo Mo(VI) centers, the 895 cm⁻¹ vibration in CODH can be assigned as the symmetric O-Mo-O stretch (ν_s) and the 861 cm⁻¹ vibration as the asymmetric O-Mo-O stretch (ν_as). Since the intensity of the assigned asymmetric O-Mo-O stretch is greater than that of ν_s, it is possible that the low symmetry of the active site, coupled with mode localization and an excited state distortion predominantly along the equatorial Mo-O_eq bond, are responsible for the nature of the unusual resonance enhancement pattern. Also, by analogy to plant sulfite oxidase,¹⁰¹ the 879 cm⁻¹ vibration may be assigned as a PDT dithiolene C-S stretch that also possesses some Mo-O_eq stretching character. The observation of multiple Mo-O_oxo stretching vibrations in the Raman spectrum of CODH is consistent with a [(PDT)MoO₂-SCu(S_Cys)]²- dioxo site for CODH_0x, and supports the mechanistic hypothesis of nucleophilic attack by the equatorial oxo ligand on the C≡O carbon atom of the substrate bound to Cu(I).^{5, 76, 130}

Figure 17.

Resonance Raman spectrum of O. carboxidovorans compared to the Raman spectra of FAD, desulfo-XO, and SO. Reproduced from ref. 68.

Conclusions

Our understanding of pyranopterin Mo enzymes has benefitted greatly from the resonance Raman spectroscopic studies detailed here. The highest information content is obtained with the use of isotope or heavy atom congener perturbations, the construction of resonance Raman excitation profiles, parallel studies on accurate structural models, and DFT frequency computations. Critical pyranopterin dithiolene Mo-S vibrational modes are now available for direct comparison between Type III DMSOR family enzymes and xanthine oxidases, indicating differences in Mo-S bonding between these two different enzymes. Raman spectroscopy has increased our current understanding of Mo-O_oxo stretching modes in the low symmetry environment of SO family sulfite oxidizing enzymes, and shows that the equatorial Mo-O_oxo bond that is directed toward the substrate access channel is specifically activated for oxygen atom transfer reactivity. The observation of Mo-product vibrational modes in DMSOR and XO have contributed greatly to our understanding of oxygen atom transfer reactivity and the unique hydroxylation mechanism posited for xanthine oxidase family enzymes. New interpretations of older Raman data have allowed for new insight into molybdoenzyme electronic structure, and this has improved our understanding of electronic structure contributions to reactivity. We expect new resonance Raman spectroscopic studies of pyranopterin Mo enzymes to further increase our understanding of enzymes beyond the canonical SO, XO, and DMSOR family enzymes that have been studied to date.

Table of Abbreviations

rR

resonance Raman

SO

sulfite oxidase

DMSOR

dimethyl sulfoxide reductase

XO

xanthine oxidase

PDT

pyranopterin dithiolene

MPT

molybdopterin

EXAFS

extended X-ray absorption fine structure

AO

aldehyde oxidase

E-P

enzyme-product

XDH

xanthine dehydrogenase

DFT

density functional theory

NBO

natural bond order

CODH

carbon monoxide dehydrogenase

Qk

normal coordinate vibration along mode q

bdt

1,2-benzenedithiolate

tdt

3,4-toluenedithiolate

qdt

quinoxaline dithiolate

Tp*

hydrotris-(3,5-dimethyl-1-pyrazolyl)borate

LMCT

ligand-to-metal charge transfer

LUMO

lowest unoccupied molecular orbital

SERR

surface enhanced resonance Raman

SAM

self-assembled monolayer

MCD

magnetic circular dichroism

EPR

electron paramagnetic resonance

BSOR

biotin sulfoxide reductase

DMS

dimethyl sulfide

NIR

near infrared

BSO

biotin sulfoxide

FAD

flavin adenine dinucleotide

MLCT

metal-to-ligand charge transfer