ADP-ribosylation of variants Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The s tructure of the dinitrogenase reductase from Azotobacter vinelandii is know n. In this study, mutant forms of dinitrogenase reductase from A. vinelandi i that are affected in various protein activities were tested for their abi lity to be ADP-ribosylated or to form a complex with dinitrogenase reductas e ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitroge nase reductase could not be ADP-ribosylated by DRAT, although it still form ed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitro genase reductase plays a critical role in the ADP-ribosylation reaction. Co nformational changes in dinitrogenase reductase induced by an F135Y substit ution or by removal of the Fe4S4 cluster resulted in dinitrogenase reductas e not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenas e reductase to form a crosslinkable complex with DRAT. Substitution of D129 E or deletion of Leu 127, which result in altered nucleotide binding region s of these dinitrogenase reductases, did not significantly change the inter action between dinitrogenase reductase and DRAT. Previous results showed th at changing Lys 143 to Gln decreased the binding between dinitrogenase redu ctase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); h owever, this change did not have a substantial effect on the interaction be tween dinitrogenase reductase and DRAT.