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Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum.

1986; Elsevier BV; Volume: 261; Issue: 11 Linguagem: Inglês

10.1016/s0021-9258(19)89201-3

1083-351X

Leonard L. Saari, Mark R. Pope, S A Murrell, Paul W. Ludden,

Enzyme Catalysis and Immobilization

Removalof ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein.A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]or [G-3ZP]ADP-ribose.The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction.Both ATP and MnClz were required for the activation of inactive iron protein.The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 m M and 2 mM for [MnClZ] and [ATP], respectively.The K,,, for inactive iron protein was 74 NM and the V , , was 628 pmol of TS2P] ADP-ribose released min-l pg of activating enzyme-1.Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein.Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP.ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.A number of microorganisms capable of nitrogen fixation are able to reversibly regulate their nitrogenase activity in response to fixed nitrogen (1-8).This phenomenon is termed "switch-off" (6) to distinguish it from regulation of the biosynthesis of the nitrogenase components.In the photosynthetic bacterium, Rhodospirillum rubrum, switch-off of nitrogenase activity is due to the ADP-ribosylation (formerly called modification) of the iron protein of nitrogenase (9-12).The inactive, ADP-ribosylated iron protein can be activated in vitro by incubation with a divalent metal ion, ATP, and an activating enzyme isolated from the chromatophore membranes of R. rubrum (13, 14).Rhodopseudomonas capsuluta (X), Azospirillum brasilense (16), and Chromatium vinosum (17) also exhibit activating enzyme activity.The expression (18), metal requirements (19-21), purification (9, 19, 22,23), and other properties (22,23) of activating