Vibrational Probes of Molybdenum Cofactor–Protein Interactions in Xanthine Dehydrogenase

The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen-bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco–protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme–product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the −NH₂ terminus of the PDT, and between Q197 and the terminal oxo of the MoO group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters <i>k</i>_red and <i>k</i>_red/<i>K</i>_D, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes

Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X‑ray Absorption Spectroscopy

Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium <i>Rhodobacter capsulatus</i>, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5–10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo­(VI) ion with each one MPT, MoO, Mo–O^–, and MoS ligand, and a weak Mo–O­(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (p<i>K</i> ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (p<i>K</i> ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic p<i>K</i> for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo–OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation

Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus

Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (<i>Rc</i>FDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO^–) to CO₂ oxidation. We characterized the molecular structure of the active site of wildtype <i>Rc</i>FDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S­(Cys386)) to Mo­(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo­(V) in a chemically reduced enzyme, and replacement of only the S­(Cys386) ligand by an oxygen of formate upon Mo­(IV) formation. The lack of a Mo=S bond in <i>Rc</i>FDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo–S­(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Mo–ligands are essential for catalysis. Low-pH inhibition of <i>Rc</i>FDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S­(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of <i>Rc</i>FDH

Effect of Exchange of the Cysteine Molybdenum Ligand with Selenocysteine on the Structure and Function of the Active Site in Human Sulfite Oxidase

Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in <i>Escherichia coli</i>. The sulfite oxidizing activity (<i>k</i>_cat/<i>K</i>_M) of SeSOMD_4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD_4Ser and SOMD_4Cys(wt). X-ray absorption spectroscopy revealed a Mo^VI–Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se­(Cys)­O₂Mo^VIS₂(MPT) site structures in the three constructs. The low-pH form of the Mo­(V) electron paramagnetic resonance (EPR) signal of SeSOMD_4Ser was altered compared to those of SOMD_4Ser and SOMD_4Cys(wt), with <i>g</i>₁ in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo­(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo–Se bond