Expression of Ni-Fe hydrogenase structural genes derived from Thiocapsa roseopersicina in Escherichia coli

Abstract

Obtaining high amounts of pure hydrogenase is a key objective, because this enzyme may be used in fuel cells and H 2 sensors. In this aspect, the HydSL Ni‐Fe hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina , which exhibits a high activity and is resistant to elevated temperatures and oxygen, is of special interest. Unfortunately, since T. roseopersicina is difficult to culture and synthesizes the HydSL Ni‐Fe hydrogenase at a very low level, the production of this enzyme in Escherichia coli would be invaluable. To study this possibility, we investigated the products of expression of structural genes encoding two subunits of the HydSL Ni‐Fe hydrogenase in this heterologous system. The major part of the protein was present in inclusion bodies, and a smaller part was in solution. Electron micrographs revealed different structure and electron density of inclusion bodies corresponding to the products of two different structural genes. We assumed that the difference in the electron density may be due to inclusion of metals in one of the subunits. Hydrogenase (EC 1.12.xx) is an enzyme catalyzing the reaction of molecular oxygen activation. Depending on the metal contained in the active site of the enzyme, all enzymes of this class are divided into Fe-containing hydrogenases, Ni‐Fe-containing hydrogenases, and hydrogenases whose reaction site contains an iron ion that is not incorporated into the protein globule but is present as a cofactor [1]. The interest to hydrogenases is determined by both active studies of the formation of the structure and the mechanism of action of complex metalloenzymes and the possibility of their use in practice in fuel cells and H 2 sensors.