Atomic resolution crystallography reveals how changes in pH shape the protein microenvironment

Hydrogen atoms are a vital component of enzyme structure and function(1-4). In recent years, atomic resolution crystallography (>= 1.2 angstrom) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis(5-9). Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.