Xanthine oxidase-product complexes probe the importance of substrate/product orientation along the reaction coordinate

Abstract

A combination of reaction coordinate computations, resonance Raman spectroscopy, spectroscopic computations, and hydrogen bonding investigations have been used to understand the importance of substrate orientation along the xanthine oxidase reaction coordinate. Specifically, 4-thiolumazine and 2,4-dithiolumazine have been used as reducing substrates for xanthine oxidase to form stable enzyme-product charge transfer complexes suitable for spectroscopic study. Laser excitation into the near-infrared molybdenum-to-product charge transfer band produces rR enhancement patterns in the high frequency in-plane stretching region that directly probe the nature of this MLCT transition and provide insight into the effects of electron redistribution along the reaction coordinate between the transition state and the stable enzyme-product intermediate, including the role of the covalent Mo-O-C linkage in facilitating this process. The results clearly show that specific Mo-substrate orientations allow for enhanced electronic coupling and facilitate strong hydrogen bonding interactions with amino acid residues in the substrate binding pocket.

Figure 1

A generalized mechanism for the oxidation of heterocyclic substrates by XO/XDH. The carbon atom that is hydroxylated is the C7 carbon for lumazine and the C8 carbon for xanthine. These C atoms and the H being transferred to the sulfido ligand are highlighted red.

Figure 2

Top: Schematic energy profile along the reaction coordinate from the IM1 Reactant state to the IM2 Product state (Mo(VI)-S to Mo(IV)-P) as a function of the C₇-H (labelled in red in Figure 1) distance (Q) of substrate (4-thiolumazine). The red arrow depicts a hypothetical two-electron transition to the Frank-Condon ES, and the blue arrow depicts the instantaneous distorting force toward the minimum along the ES PES. Bottom: Simple diagram showing how two electrons are transferred from the Substrate HOMO to the Mo(xy) redox orbital along the reaction coordinate. In this two-state frontier orbital depiction, the Substrate HOMO smoothly transitions to the Product LUMO.

Figure 3

Top: Computed HOMO and LUMO wavefunctinos for the XOr-4-TV E-P complex. Bottom: Electron density difference map (EDDM) for the HOMO → LUMO MLCT transition. Purple represents an electron density loss in the transition and orange represents an electron density gain. The view is oriented looking down the Mo≡O bond.

Figure 4

Experimental (red) and computed (black) resonance Raman spectra of XOr-4-TV (top) and XOr-2,4-TV (bottom) collected anaerobically in BICINE buffer (pH=8.3). The asterisk is from (NH₄)₂SO₄ in the buffer and serves as an internal standard.

Figure 5

Selected computed vibrational modes for XOr-4-TV complex, which are rendered and superimposed at the minimum and maximum values of the vibrational coordinate, Q, during the vibrational period.

Figure 6

Top: “Upside” and “Upside down” orientations of 4-thiolumazine in IM1. Bottom: “Upside” orientation with protonated E802. (I changed to E802. E232 Should be E802 for XO if use R880; or for XDH they are E232 and R310.)

Figure 7

ORTEP view of the hydrogen-bonded 4-thiolumazine●DMF dimer showing the thermal ellipsoids at 50% probability. Hydrogen atoms are fixed as 0.15 Å sphere. Crystal parameters: monoclinic P1/c. a) 5.566(3), b) 8.588(4), c)12.217(7) Å; α) 97.751(4)°, β) 98.795(3)°, γ) 101.942(3)°. Hydrogen bonding distance: N3H3—N2: 2.086(2) Å; N4H4—O2: 1.879(2) Å.

Figure 8

Gas-phase optimized geometries for (A) the “upside” orientation and (B) the “upside down” orientation of 4-TV in the reduced Mo(IV)-P complex. Three conserved amino acids have been included in the calculation. Note that the “upside” orientation stabilizes the Mo(IV)-P complex by 14.5 kcal/mol relative to the “upside down” orientation.