Chloroperoxidase, a heme-thiolate protein with exceptional versatility, contains 2 amide moieties which form strong hydrogen bonds with the sulfur (NH•••S hydrogen bonds) of the proximal cysteine thiolate ligand. This ligand has been the subject of many studies and has been proposed to weaken the O-O bond of the ferric hydroperoxide intermediate, Compound 0 (Cpd 0) via the “push effect”  by serving as a strong electron donor, “pushing” electrons to facilitate heterolytic O-O bond scission to form Cpd I. The role of the NH•••S hydrogen bonds has been significantly debated, as their suggested function has ranged from passive (protecting the axial sulfur) to active (modulating O-O bond scission in Cpd 0). In order to determine how influential these hydrogen bonds are on the mechanism of Cpd I formation, we have built 2 comparative model systems (one with and one without a proximal region) which serve to isolate the influence of the hydrogen bonding interaction. Reaction profiles for the transformation of the ferric resting state into Compound I for both models were obtained using DFT calculations. We find that NH•••S hydrogen bonds: (1) reduce the barrier for the formation of Compound 0 by 7.7 kcal/mol, (2) increase the stability of Compound 0 by 5.4 kcal/mol, (3) reduce the Compound 0/Compound I relative stability by 7.0 kcal/mol, and (4) increase the barrier for the transformation of Compound 0 into Compound I by 3.8 kcal/mol. The NH•••S hydrogen bonds also reduce the stability of a protonated Compound 0 species, increasing the favorability of a proton-coupled electron transfer mechanism, relative to a classic heterolytic mechanism for O-O bond scission. In general, the influence of the NH•••S hydrogen bonds can be traced to a reduction in the pKa of the distal ligand. We find that the hydrogen bond networks on the proximal and distal sides of the heme function together to modulate the mechanism of reaction, as suggested by Poulos. These results confirm and extend long-standing theories that the NH•••S hydrogen bonds in heme thiolate proteins influence reactivity.

DFT study elucidates proximal pocket hydrogen bond influence on mechanism of compound I formation in chloroperoxidase

249th National Meeting (2015)

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