Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe4S4](1+/2+) cluster of nitrogenase Fe protein from R, rubrum or Azotobacter vinelandii, DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein, The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N-alpha-dansyl-N-omega-(1,N-6-etheno-ADP-ribosyl) arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe4S4] cluster from R, rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<2%) activity with 87% reduced R, rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mw NH4Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.