Three genetically distinct, but structurally similar, isozymes of nitrogenase catalyze biological N2 reduction to 2NH3: Mo-, V-, and Fe-nitrogenase, named respectively for the metal (M) in their active site metallocofactors (metal-ion composition, MFe7). Studies of the Mo-enzyme have revealed key aspects of its mechanism for N2 binding and reduction. Central to this mechanism is accumulation of four electrons and protons on its active site metallocofactor, called FeMo-co, as metal bound hydrides to generate the key E4(4H) (“Janus”) state. N2 binding/reduction in this state is coupled to reductive elimination (re) of the two hydrides as H2, the forward direction of a reductive-elimination/oxidative-addition (re/oa) equilibrium. A recent study demonstrated that Fe-nitrogenase follows the same re/oa mechanism, as particularly evidenced by HD formation during turnover under N2/D2. Kinetic analysis revealed that Mo- and Fe-nitrogenases show similar rate constants for hydrogenase-like H2 formation by hydride protonolysis (k HP) but significant differences in the rate constant for H2 re with N2 binding/reduction (k re ). We now report that V-nitrogenase also exhibits HD formation during N2/D2 turnover (and H2 inhibition of N2 reduction), thereby establishing the re/oa equilibrium as a universal mechanism for N2 binding and activation among the three nitrogenases. Kinetic analysis further reveals that differences in catalytic efficiencies do not stem from significant differences in the rate constant (k HP) for H2 production by the hydrogenase-like side reaction but directly arise from the differences in the rate constant (k re ) for the re of H2 coupled to N2 binding/reduction, which decreases in the order Mo > V > Fe.

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2...

Coupling the nitrogenase MoFe protein to light-harvesting semiconductor nanomaterials replaces the natural electron transfer complex of Fe protein and ATP and provides low-potential photoexcited electrons for photocatalytic N 2 reduction. A central question is how direct photochemical electron delivery from nanocrystals to MoFe protein is able to support the multielectron ammonia production reaction. In this study, low photon flux conditions were used to identify the initial reaction intermediates of CdS quantum dot (QD):MoFe protein nitrogenase complexes under photochemical activation using EPR. Illumination of CdS QD:MoFe protein complexes led to redox changes in the MoFe protein active site FeMo-co observed as the gradual decline in the E 0 resting state intensity that was accompanied by an increase in the intensity of a new "g eff = 4.5" EPR signal. The magnetic properties of the g eff = 4.5 signal support assignment as a reduced S = 3/2 state, and reaction modeling was used to define it as a two-electron-reduced "E 2 " intermediate. Use of a MoFe protein variant, β-188 Cys , which poises the P cluster in the oxidized P + state, demonstrated that the P cluster can function as a site of photoexcited electron delivery from CdS to MoFe protein. Overall, the results establish the initial steps for how photoexcited CdS delivers electrons into the MoFe protein during reduction of N 2 to ammonia and the role of electron flux in the photochemical reaction cycle.

The reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase MoFe protein is coupled to chemically driven electron transfer by nitrogenase Fe protein, where H2 is an obligatory side product. Direct coupling of light-absorbing semiconductor nanocrystals to MoFe protein enables NH3 production from photoexcited electron transfer, replacing Fe protein. Production of H2 and NH3 was measured for CdS quantum dot (QD) MoFe protein complexes illuminated under different excitation rates. 15N-labeling of NH3 production combined with background-corrected H2 production enabled determination of MoFe protein catalysis products. The turnover rates of H2 and NH3 increased with excitation rate, with distinct kinetic responses that show the electron demand for NH3 requires higher excitation rates to overcome the more favored H2 production.