Purification and characterization of ferredoxins from the heliobacterium Heliobacillus mobilis

Abstract

The reaction center (RC) of heliobacteria belongs to an iron sulfur type, and the electron transfer pathway around the RC remains uncertain. To study the electron pathway in heliobacteria from RC to ferredoxin (Fd) and to further electron acceptors, we have isolated and purified Fds from the heliobacterium Heliobacillus mobilis. Two Fds, termed Fd-A and Fd-B, were isolated from Heliobacillus mobilis cells, and purified by ammonium sulfate fractionation, DEAE and Phenyl-Superose column chromatographies under anaerobic conditions. Their absorption spectra were typical of 2[4Fe-4S] center with absorption peaks at 385 and 280 nm and with a shoulder at 305 nm. Fd-B was sensitive to oxygen, and its A385 values decreased by about 50% in 2 hours at 4oC under aerobic conditions. In contrast with this, A385 of Fd-A was unchanged up to 24 h under the same conditions. In the presence of Fd-NADP+ reductase from spinach and purified Fe-S type reaction center from the green sulfur bacterium Chlorobium tepidum, these Fds supported photoreduction of NADP+ at room temperature. Cryogenic EPR measurements indicated that Fd-A has EPR signals at g = about 2.05, 1.94, and Fd-B at g = about 2.05, 1.92, respectively. Both Fds were fully reduced by dithionite at pH 8.0.