Zinc-, cobalt- and iron-chelated forms of adenylate kinase from the Gram-negative bacterium Desulfovibrio gigas

Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co2+/Zn2+-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co2+-, Zn2+- and Fe2+-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co2+- and Zn2+-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe2+-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe2+-AK, in contrast to Co2+- and Zn2+-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.