Computational Insights into Sulphur-Crosslinking Enzymatic Mechanisms

Sulfur plays a diverse array of important roles in biochemical systems. This is due to its ability to possess a range of oxidation states, engage in a variety of bonding environments (e.g., thiols, disulfide bonds, hypervalent species), and reversibly participate in redox chemistry. In this thesis we have examined two biochemically important system that involve sulfur and exploit its rich chemistry. In Chapter 3 we examine the hypobromous acid mediated formation of the ancient but critical inter-protein sulfilimine (S=N) crosslink of Collagen IV. Previously it was proposed to form either via a halosulfonium or haloamine intermediate, with the pathway via the former being preferred. Using a density functional theory-chemical cluster (QM-cluster) approach we show that formation of a haloamine intermediate is thermodynamically favoured, with it lying much lower in energy than the proposed halsulfonium intermediate. However, its barrier to formation is higher than for the halosulfonium intermediate. Hence, formation of the sulfilimine bond via the latter is kinetically favoured. In chapter 4, we examine possible mechanisms by which the iron-dependent enzyme thiazole synthase forms a life-essential thiazole ring-containing metabolite. In particular, comparison of available experimental crystal structures with QM-cluster and QM/MM optimized enzyme-substrate structures shows that the substrate is unlikely to be a glycyl-imine. Rather, it is more likely a -C(CH3)=N- containing species. Furthermore, water density analysis of the substrate-bound active site suggests that it may be the conduit for mechanistically required protons to enter the active site. In addition, our results suggest that the loss of the iron ion from the active site may occur earlier in the mechanism than proposed, before formation of the proposed thione intermediate. These results provide new insights into these important bonds and species and lay the ground work for further computational and experimental studies