Mechanistic insights into the chemistry of compound I formation in heme peroxidases: quantum chemical investigations of cytochrome c peroxidase

Abstract

Peroxidases are heme containing enzymes that catalyze peroxide-dependant oxidation of a variety of substrates through forming key ferryl intermediates, compounds I and II. Cytochrome c peroxidase (Ccp1) has served for decades as a chemical model toward understanding the chemical biology of this heme family of enzymes. It is known to feature a distinctive electronic behaviour for its compound I despite significant structural similarity to other peroxidases. A water-assisted mechanism has been proposed over a dry one for the formation of compound I in similar peroxidases. To better identify the viability of these mechanisms, we employed quantum chemistry calculations for the heme pocket of Ccp1 in three different spin states. We provided comparative energetic and structural results for the six possible pathways that suggest the preference of the dry mechanism energetically and structurally. The doublet state is found to be the most preferable spin state for the mechanism to proceed and for the formation of the Cpd I ferryl-intermediate irrespective of the considered dielectric constant used to represent the solvent environment. The nature of the spin state has negligible effects on the calculated structures but great impact on the energetics. Our analysis was also expanded to explain the major contribution of key residues to the peroxidase activity of Ccp1 through exploring the mechanism at various in silico generated Ccp1 variants. Overall, we provide valuable findings toward solving the current ambiguity of the exact mechanism in Ccp1, which could be applied to peroxidases with similar heme pockets.

Scheme 1. Proposed catalytic mechanism of Cpd I formation in heme peroxidases.

Fig. 1. Cluster structures along the wet-mechanism pathway of Cpd I formation, with selected distances between heavy atoms (Å), for three different spin states: doublet (black), quartet (blue) and sextet (red). Geometries are optimized with UB3LYP-D3BJ/6-31G(d,p).

Fig. 2. Cluster structures along the dry-mechanism pathway of Cpd I formation, with selected distances between heavy atoms (Å), for three different spin states: doublet (black), quartet (blue) and sextet (red). Geometries are optimized with UB3LYP-D3BJ/6-31G(d,p).

Fig. 3. Free-energy profiles (kcal mol⁻¹) for both the wet (a), and dry (b) mechanisms of Cpd I formation for three different spin states: doublet (black line), quartet (blue line) and sextet (red line). Free energies are estimated with UB3LYP-GD3BJ/6-311+G(2d,2p) with ε = 4 (see text for details).

Fig. 4. Spin density surfaces together with values for the three major spin contaminated components of (A) doublet (B) quartet (C) sextet. Calculated with UB3LYP-D3BJ/6-31G(d,p).

Fig. 5. Spin density surfaces together with values for the three major spin contaminated components of WT Ccp1 and its in silico generated variants. Calculated with UB3LYP-D3BJ/6-31G(d,p).