Metabolic regulation of nitrogenase in Rhodospirillum rubrum : studies on DRAG and DRAT, the regulatory proteins of dinitrogenase reductase

• Nitrogen fixation is catalyzed by nitrogenase, in an ATP-dependent reaction where dinitrogen is reduced to ammonium. Nitrogenase is a protein complex consisting of two proteins; dinitrogenase and dinitrogenase reductase and the reaction is only carried out by prokaryotes. In the photosynthetic bacterium, Rhodospirillum rubrum, nitrogen fixation is regulated at both a genetic and a metabolic level. The metabolic regulation is excerted through post-translational modification of dinitrogenase reductase, causing inactivation of the enzyme when so called switch-off effectors are added. The post-translational modification has been shown to be an ADP-ribosylation on Arg-101 of dinitrogenase reductase. The regulatory proteins are; dinitrogenase reductase ADP-ribosyl transferase (DRAT), catalyzing modification and dinitrogenase reductase activating glycohydrolase (DRAG) catalyzing demodification. Both DRAG and DRAT are subjected to post-translational regulation in the cell. The internal signals for this regulation are still not identified and have been the focus of this thesis.By changing the concentration of NAD+ in R.rubrum a reversible decrease in nitrogenase activity could be observed. This decrease in activity was shown to be correlated to a post-translational modification of dinitrogenase reductase by SDS-PAGE and Western blot. The modification is catalyzed by DRAT since studies on a DRAT- mutant show no response at all to additional NAD+. Shortening the switch-off period by addition of NADH generating compounds, confirmed that a change in NAD+ /NADH ratio affects the duration of the switch-off period. Using in vivo fluorometry to study the effect of changes in the NAD+ concentration when nitrogenous switch-off effectors were added, showed that ammonium and glutamine momentarily change the concentration of NAD+. On the basis of these results we suggest that the signal pathway for DRAT activation involves changes in the concentration of NAD+.The membrane associated DRAG is shown to be released by treating the chromatophores with MgGDP, similar treatment with MgGTP, MgATP or MgADP did not result in such a release. Upto 40% of DRAG activity can be found in the supernatant compared to 0.5 M NaCl treated membranes. The chromatophore membranes are shown to have GTPase activity with and without DRAG associated to the membranes indicating the presence of a GTP hydrolyzing protein in the chromatophore membrane. Crosslinking studies demonstrated a high molecular weight complex, that crossreacted with DRAG antibodies.The regulation of DRAG is suggested by us to be mediated by the chromatophore membrane possibly involving a protein complex with GTPase activity. DRAG is suggested to be active in its soluble form and inactive when bound to the membranes.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Biochemistry

Biokemi

Biochemistry

biokemi

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