Mechanism of Mo-dependent nitrogenase

Abstract

Nitrogen-fixing bacteria catalyze the reduction of dinitrogen (N(2)) to two ammonia molecules (NH(3)), the major contribution of fixed nitrogen to the biogeochemical nitrogen cycle. The most widely studied nitrogenase is the molybdenum (Mo)-dependent enzyme. The reduction of N(2) by this enzyme involves the transient interaction of two component proteins, designated the iron (Fe) protein and the MoFe protein, and minimally requires 16 magnesium ATP (MgATP), eight protons, and eight electrons. The current state of knowledge on how these proteins and small molecules together effect the reduction of N(2) to ammonia is reviewed. Included is a summary of the roles of the Fe protein and MgATP hydrolysis, information on the roles of the two metal clusters contained in the MoFe protein in catalysis, insights gained from recent success in trapping substrates and inhibitors at the active-site metal cluster FeMo cofactor, and finally, considerations of the mechanism of N(2) reduction catalyzed by nitrogenase.

Figure 1

Structures of the nitrogenase MoFe and Fe proteins. The MoFe protein is an α₂β₂ tetramer, with the alpha subunits shown in magenta and the beta subunits shown in green. The Fe protein is a γ₂ dimer, with each subunit shown in blue. A MoFe protein binds two Fe proteins, with each αβ unit being a catalytic unit. One Fe protein is shown associating with one αβ-unit of the MoFe protein. The relative positions and structures of two bound MgADP molecules, the Fe protein [4Fe-4S] cluster, and MoFe protein P-cluster (8Fe-7S), and FeMo-cofactor (7Fe-Mo-9S-homocitrate-X) are shown. Each is highlighted to the right. The flow of electrons is from the [4Fe-4S] cluster to the P-cluster to the FeMo-cofactor. The element color scheme is C in gray, O is red, N in blue, Fe in rust, S in yellow, and Mo in magenta. Graphics were generated with the program Pymol using the Protein Data Base (PDB) files 1M1N for the MoFe protein and 1FP6 for the Fe protein.

Figure 2

Fe and MoFe protein catalytic cycles. Shown is a three state cycle for the Fe protein (top) and an eight state cycle for the MoFe protein (bottom). For the Fe protein (abbreviated FeP), the [4Fe-4S] cluster can exist in the +1 reduced state (Red) or the 2+ oxidized state (Ox). The Fe protein either has two MgATP molecules bound (ATP) or two MgADP with two Pi (ADP+Pi). The exchange of an electron occurs upon association of the Fe protein with the MoFe protein at the bottom of the cycle. In the MoFe protein cycle, the MoFe protein is successively reduced by one electron, with reduced states represented by E_n, where n is the total number of electrons donated by the Fe protein. Acetylene (C₂H₂) is shown binding to E₂, while N₂ is shown binding to E₃ and E₄. N₂ binding is accompanied by the displacement of H₂. The two ammonia molecules are shown being liberated from later E states.

Figure 3

Structure of the FeMo-cofactor of nitrogenase. The element colors are as described in the legend to Figure 1.

Figure 4

P-cluster structures. Shown are the structures of the P-cluster [8Fe-7S] in the oxidized (P^ox) and reduced (P^N) states. MoFe protein amino acid ligands are also shown with β-188^Ser and α-88^Cys labeled. The central S atom is labeled S1. The PBD files used were 2MIN for the P^ox state and 3MIN for the P^N state.

Figure 5

Substrates for nitrogenase. Shown are the structures for select nitrogenase substrates. Compounds that are substrates for the wild-type enzyme are shown to the left, while compounds that become substrate for MoFe proteins with amino acid substitutions for α-70^Val are shown to the right.

Figure 6

Substrate binding location on FeMo-cofactor. Shown is the FeMo-cofactor with Fe atoms 2, 3, 6, and 7 labeled. The view is from the top looking down on the Fe face that binds substrates. Carbon alpha and the side chain are shown for α-69^Gly, α-70^Val, α-195^His, and α-191^Gln. PDB file 1M1N.

Figure 7

Control of substrate access to FeMo-cofactor. Shown is the FeMo-cofactor without R-homocitrate viewed down the Mo end. The side chain of α-70^Val is shown with a Van der Waals mesh (left). Also shown are computer generated models of the Van der Waals surface for α-70^Ala (center) and α-70^Ile (right) substituted MoFe proteins.

Figure 8

EPR spectra of nitrogenase with trapped substrates. Shown are EPR spectra for various MoFe protein variants that were frozen in liquid nitrogen either in the resting state (top spectrum) or during steady state turnover (all other spectra) in the presence of different substrates. The MoFe protein and substrate is noted on each spectrum. EPR spectra were acquired between 8–12 K.

Figure 9

Possible reaction mechanisms for nitrogenase. Shown are two possible reaction mechanisms for nitrogenase. On the left is shown the distal mechanism and on the right the alternating mechanism. FeMo-cofactor is abbreviated as M and the names of different bound states are shown. Possible points of entry for diazene and hydrazine are shown.