Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties.

Abstract

Nitrogenase proteins were isolated from cultures of the photosynthetic bacterium Rhodopseudomonas capsulata grown on a limiting amount of ammonia. Under these conditions, the nitrogenase N2ase A was active in vivo, and nitrogenase activity in vitro was not dependent upon manganese and the activating factor. The nitrogenase proteins were also isolated from nitrogen-limited cultures in which the in vivo nitrogenase activity had been stopped by an ammonia shock. This nitrogenase activity, N2ase R, showed an in vitro requirement for manganese and the activating factor for maximal activity. The Mo-Fe protein (dinitrogenase) was composed of two dissimilar subunits with molecular weights of 55,000 and 59,500; the Fe protein (dinitrogenase reductase), from either type of culture, was composed of a single subunit (molecular weight, 33,500). The metal and acid labile sulfur contents of both nitrogenase proteins were similar to those found for previously isolated nitrogenases. The Fe proteins from both N2ase A and N2ase R contained phosphate and ribose, 2 mol of each per mol of N2ase R Fe protein and about 1 mol of each per mol of N2ase A Fe protein. The greatest difference between the two types of Fe protein was that the N2ase R Fe protein contained about 1 mol per mol of an adenine-like molecule, whereas the N2ase A Fe protein content of this compound was insignificant. These results are compared with various models previously presented for the short-term regulation of nitrogenase activity in the photosynthetic bacteria. 708 on July 31, 2020 by guest

Biochemical genetics revisited: the use of mutants to study carbon and nitrogen metabolism in the photosynthetic bacteria

“…rubrum and Rb. capsulatus [172][173][174]. In both species, nitrogenase is subject to reversible inactivation or 'switch-off by NH~-, due to covalent modification of the iron protein.…”

Regulation of nitrogenase activity inAnabaena variabilisby modification of the Fe protein

“…Ceil-free nitrogenase activity was measured with 5 mM sodium dithionite as electron donor and with an ATP-regenerating system present as described [9]. In the case of partially purified components 1 mg BSA × nag -1 was added and the two components (Fe and Mo-Fe protein) were titrated to obtain maximum acetylene-reduction rates [17].…”

Purification of a sixth ferredoxin from Rhodobacter capsulatus

“…It can grow autotrophically or heterotrophically either in the light or in darkness and can choose between five different growth modes (Madigan and Gest, 1979). It is also capable of fixing molecular nitrogen through a metabolic process which has been extensively studied by biochemical (Hallenbeck et al, 1982a; and genetic approaches (Willison et al, 1985;Klipp et al, 1988). Nitrogen fixation is catalyzed by nitrogenase and requires ATP and a low-potential reductant.…”