Nimesh Khadka

Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.

Enzymes make fertilizer with sunlight Nitrogenase enzymes catalyze the biological production of fixed nitrogen. Because this is not enough to sustain modern agriculture, industrial fertilizers containing ammonia are produced via the energy-intensive Haber-Bosch process. Brown et al. developed a way to use nitrogenase enzymes from nitrogen-fixing bacteria to make ammonia in vitro without other biological steps or high-energy inputs. Light-activated CdS nanorods provided electrons to the FeMo nitrogenase enzyme to reduce nitrogen and produce ammonia at rates up to 64% of biological nitrogen fixation. These nanoparticle-protein complexes show the potential for solar-driven ammonia production. Science, this issue p. 448 Enzymes attached to photoexcited semiconductor nanorods turn nitrogen into ammonia. The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5′-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3. The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

On reversible H2 loss upon N2 binding to FeMo-cofactor of nitrogenase

Significance Biological reduction of dinitrogen (N2) to two ammonia (NH3) is catalyzed by the metalloenzyme, nitrogenase. A central aspect of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, with a resulting “waste” of the energy required to deliver two electrons/protons, obtained from hydrolysis of four ATP. We here report experiments that confirm the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, not six as in the equation N2 + 3H2 → 2NH3. These experiments were devised to test our recently proposed “reductive elimination” mechanism for H2 formation and the activation of nitrogenase for ammonia production. Our findings provide direct experimental support for that mechanism. Nitrogenase is activated for N2 reduction by the accumulation of four electrons/protons on its active site FeMo-cofactor, yielding a state, designated as E4, which contains two iron-bridging hydrides [Fe–H–Fe]. A central puzzle of nitrogenase function is an apparently obligatory formation of one H2 per N2 reduced, which would “waste” two reducing equivalents and four ATP. We recently presented a draft mechanism for nitrogenase that provides an explanation for obligatory H2 production. In this model, H2 is produced by reductive elimination of the two bridging hydrides of E4 during N2 binding. This process releases H2, yielding N2 bound to FeMo-cofactor that is doubly reduced relative to the resting redox level, and thereby is activated to promptly generate bound diazene (HN=NH). This mechanism predicts that during turnover under D2/N2, the reverse reaction of D2 with the N2-bound product of reductive elimination would generate dideutero-E4 [E4(2D)], which can relax with loss of HD to the state designated E2, with a single deuteride bridge [E2(D)]. Neither of these deuterated intermediate states could otherwise form in H2O buffer. The predicted E2(D) and E4(2D) states are here established by intercepting them with the nonphysiological substrate acetylene (C2H2) to generate deuterated ethylenes (C2H3D and C2H2D2). The demonstration that gaseous H2/D2 can reduce a substrate other than H+ with N2 as a cocatalyst confirms the essential mechanistic role for H2 formation, and hence a limiting stoichiometry for biological nitrogen fixation of eight electrons/protons, and provides direct experimental support for the reductive elimination mechanism.

Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N2 for H2

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During -50 °C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e(-)/H(+)] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3.

Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme

We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology.

Photoinduced Reductive Elimination of H2 from the Nitrogenase Dihydride (Janus) State Involves a FeMo-cofactor-H2 Intermediate

N2 reduction by nitrogenase involves the accumulation of four reducing equivalents at the active site FeMo-cofactor to form a state with two [Fe-H-Fe] bridging hydrides (denoted E4(4H), the Janus intermediate), and we recently demonstrated that the enzyme is activated to cleave the N≡N triple bond by the reductive elimination (re) of H2 from this state. We are exploring a photochemical approach to obtaining atomic-level details of the re activation process. We have shown that, when E4(4H) at cryogenic temperatures is subjected to 450 nm irradiation in an EPR cavity, it cleanly undergoes photoinduced re of H2 to give a reactive doubly reduced intermediate, denoted E4(2H)*, which corresponds to the intermediate that would form if thermal dissociative re loss of H2 preceded N2 binding. Experiments reported here establish that photoinduced re primarily occurs in two steps. Photolysis of E4(4H) generates an intermediate state that undergoes subsequent photoinduced conversion to [E4(2H)* + H2]. The experiments, supported by DFT calculations, indicate that the trapped intermediate is an H2 complex on the ground adiabatic potential energy suface that connects E4(4H) with [E4(2H)* + H2]. We suggest that this complex, denoted E4(H2; 2H), is a thermally populated intermediate in the catalytically central re of H2 by E4(4H) and that N2 reacts with this complex to complete the activated conversion of [E4(4H) + N2] into [E4(2N2H) + H2].

Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate-limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.

Cluster-dependent charge-transfer dynamics in iron-sulfur proteins.

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.