Abstract
The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α(2)ß(2)(γ(2)) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N(2) to NH(3), with no significant difference in selectivity over obligatory/parasitic H(2) formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H(2) and NH(3) formation (under Ar or N(2)), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N(2) fixation in Azotobacter vinelandii.