Structural Characterization of the Novel and Thermal Stable Hydrogenases from the Purple Sulfur Bacteria Thiocapsa Roseopersicina and Lamprobacter Modestohalophilus

摘要

This report results from a contract tasking Institute of Basic Biological Problems of RAS as follows: The hydrogenase is a key enzyme of the hydrogen metabolism of different microorganisms, catalyzing the reversible activation of molecular hydrogen. The use of this enzyme in biotechnology is often limited by its low thermostability and sensitivity to oxygen and other toxic agents. We have found that phototrophic sulfur bacteria possess very stable hydrogenases which can be used for the development of fuel cells and various photocatalytic systems for H2 production. The overall goal of the project was the biochemical and structural characterization of hydrogenases isolated and purified from the cells of the noted above phototrophic bacteria. The specific aims included: (i) the cultivation of T. roseopersicina and L. modesohalophilus in photobioreactors under optimal conditions for high output of biomass; (ii) the development of effective methods for hydrogenase purification and biochemical and catalytic characterization; (iii) obtaining the hydrogenase crystals and study of their structure. The main results obtained in the course of the Project where: 1) a large-scale cultivation of T. roseopersicina, L. modestohalophilus and Ch. aurantiacus and isolation of hydrogenase from these phototrophic bacteria allowing to obtain homogeneous preparations of the enzyme with a yield of 40-50% of the activity are developed and optimized; 2) it was discovered by the method of cryo-electron microscopy that a molecules of hydrogenase from T. roseopersicina form a ring-shaped hexameric complex with D3 symmetry. It is assumed that the formation of such hexameric structures provides increased stability of the enzyme to the action of temperature and proteases; 3) it is shown that hydrogenase T. roseopersicina exhibits high stability in the buffer systems, in which its molecules have a negative charge: at the pH range 7-9 and low ionic strength.