Solubilization of reaction center of Heliobacterium modesticaldum and its ferredoxin and NADP⁺ reduction activities

Abstract

We have prepared 4Fe-4S type ferredoxin (Fd) from the cell extract of H. modesticaldum by ammonium sulfate fractionation and anion-exchange chromatography. Crude RC-rich fractions were prepared under anaerobic conditions by solubilization with detergents of the membranes of the bacterium, and one of them showed NADP⁺ photoreduction activity in the presence of Fd I from H. modesticaldum and Fd-NADP⁺ reductase (FNR) from spinach. The active fractions partially purified by sucrose-density gradient ultracentrifugation reduced NADP⁺ at 12 μmole μmole BChl^-1 hr^-1. The peptides in the active fractions were analyzed by SDS-PAGE and TOF-Mass spectrometer. Although the whole genome sequence of this bacterium has been elucidated, the gene encoding FNR has not yet been identified. From the cell extract, we obtained several partially purified fractions containing NADPH-DPIP diaphorase activity, but the major active fraction has no FNR activity.