QM/MM study of the reaction mechanism of sulfite oxidase

Abstract

Sulfite oxidase is a mononuclear molybdenum enzyme that oxidises sulfite to sulfate in many organisms, including man. Three different reaction mechanisms have been suggested, based on experimental and computational studies. Here, we study all three with combined quantum mechanical (QM) and molecular mechanical (QM/MM) methods, including calculations with large basis sets, very large QM regions (803 atoms) and QM/MM free-energy perturbations. Our results show that the enzyme is set up to follow a mechanism in which the sulfur atom of the sulfite substrate reacts directly with the equatorial oxo ligand of the Mo ion, forming a Mo-bound sulfate product, which dissociates in the second step. The first step is rate limiting, with a barrier of 39–49 kJ/mol. The low barrier is obtained by an intricate hydrogen-bond network around the substrate, which is preserved during the reaction. This network favours the deprotonated substrate and disfavours the other two reaction mechanisms. We have studied the reaction with both an oxidised and a reduced form of the molybdopterin ligand and quantum-refinement calculations indicate that it is in the normal reduced tetrahydro form in this protein.

Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement

“…w MM is a weighting factor needed because the empirical potential of crystallographic refinement software is normally based on a statistical analysis of high-resolution crystal structures [37], rather than on energetic consideration (as for the QM term). We have shown that quantum refinement can locally improve crystal structures [31], decide protonation state of metal-bound ligands [38][39][40][41], oxidation state of metal sites [42,43] and protein ligands [41], detect photoreduction of metal ions [42,44,45] and decide what is really seen in crystal structures [44][45][46].…”

On the Difference Between Additive and Subtractive QM/MM Calculations

“…sulfite oxidase. Based on our recent QM-cluster (Van Severen et al, 2014 ) and QM/MM (Caldararu et al, 2018 ) studies, we considered the S → OMo mechanism, in which the S atom of the sulfite substrate attacks the equatorial oxo group of Mo VI , directly forming a Mo IV -sulfate intermediate (Im), via a first transition state TS1 (Figure 2 ). The sulfate product then dissociates into the second coordination sphere of the Mo ion via a second transition state (TS2).…”

“…The second test system was sulfite oxidase. The calculations were taken from our recent study of this enzyme (Caldararu et al, 2018 ). The active site contains a Mo ion coordinated to a molybdopterin (MPT) molecule, as well as a Cys residue and two oxo groups.…”

“…The active site contains a Mo ion coordinated to a molybdopterin (MPT) molecule, as well as a Cys residue and two oxo groups. All these groups were included in the QM system (Cys modeled as CH 3 S − ), as well as the sulfite substrate [note that this QM system is smaller than in our previous study (Caldararu et al, 2018 ), in which nine additional residues and five water molecules were also included]. In this paper we compared the effects of either including the full MPT residue (in the reduced and protonated form, called MPH in our previous paper) or truncating it to a dimethyldithiolene molecule [DMDT, (CH 3 CS ; both shown in Figure 2 ].…”

“…The transition states were obtained from potential-energy scans along the S–O1 (TS1, 2.0 Å) and Mo–O1 (TS2, 3.7 Å) reaction coordinates. PS was also obtained with a restraint in the Mo–O2 distance of 3.85 Å, taken from calculations with an appreciably larger QM system (Caldararu et al, 2018 ). Besides the QM system, the setup was identical to that in our previous study (Caldararu et al, 2018 ).…”