The substrate-reducing
proteins of all nitrogenases (MoFe, VFe,
and FeFe) are organized as α2ß2(γ2) multimers with two functional halves. While their dimeric
organization could afford improved structural stability of nitrogenasesin 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 N2to NH3, with no significant difference in selectivity over obligatory/parasitic
H2formation. Our experiment concludes that wild-type nitrogenase
exhibits negative cooperativity during the steady state regarding
H2and NH3formation (under Ar or N2), 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 N2fixation inAzotobacter vinelandii.