thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase.

In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQ), which is in equilibrium with a semiquinone radical (TPQ). The transition from TPQ to TPQ is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and glue-coating (HAG) method to capture the equilibrium mixture of TPQ and TPQ in noncryocooled crystals of the enzyme from and found that the equilibrium shifts more toward TPQ in crystals than in solution. Thermodynamic analyses of the temperature-dependent equilibrium also revealed that the transition to TPQ is entropy-driven both in crystals and in solution, giving the thermodynamic parameters that led to experimental determination of the crystal packing effect. Furthermore, we demonstrate that the binding of product aldehyde to the hydrophobic pocket in the active site produces various equilibrium states among two forms of the product Schiff-base, TPQ, and TPQ, in a pH-dependent manner. The temperature-controlled HAG method provides a technique for thermodynamic analysis of conformational changes occurring in protein crystals that are hardly scrutinized by conventional cryogenic X-ray crystallography.