The Fe Protein Cycle Associated with Nitrogenase Catalysis Requires the Hydrolysis of Two ATP for Each Single Electron Transfer Event.

A central feature of the current understanding of dinitrogen (N2) reduction by the enzyme nitrogenase is the proposed coupling of the hydrolysis of two ATP, forming two ADP and two Pi, to the transfer of one electron from the Fe protein component to the MoFe protein component, where substrates are reduced. A redox-active [4Fe-4S] cluster associated with the Fe protein is the agent of electron delivery, and it is well known to have a capacity to cycle between a one-electron-reduced [4Fe-4S]1+ state and an oxidized [4Fe-4S]2+ state. Recently, however, it has been shown that certain reducing agents can be used to further reduce the Fe protein [4Fe-4S] cluster to a super-reduced, all-ferrous [4Fe-4S]0 state that can be either diamagnetic (S = 0) or paramagnetic (S = 4). It has been proposed that the super-reduced state might fundamentally alter the existing model for nitrogenase energy utilization by the transfer of two electrons per Fe protein cycle linked to hydrolysis of only two ATP molecules. Here, we measure the number of ATP consumed for each electron transfer under steady-state catalysis while the Fe protein cluster is in the [4Fe-4S]1+ state and when it is in the [4Fe-4S]0 state. Both oxidation states of the Fe protein are found to operate by hydrolyzing two ATP for each single-electron transfer event. Thus, regardless of its initial redox state, the Fe protein transfers only one electron at a time to the MoFe protein in a process that requires the hydrolysis of two ATP.

Mechanistic Insights into Nitrogenase FeMo-Cofactor Catalysis through a Steady-State Kinetic Model.

Mo-nitrogenase catalyzes the challenging N2-to-NH3 reduction. This complex reaction proceeds through a series of intermediate states (En) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between En states is central to defining the mechanism of nitrogenase. Here, rate constants of key steps have been determined through a steady-state kinetic model with fits to experimental data. The model reveals that the rate for H2 formation from the early electron populated state E2(2H) is much slower than that from the more reduced E4(4H) state. Further, it is found that the competing reactions of H2 formation and N2 binding at the E4(4H) state occur with equal rate constants. The H2-dependent reverse reaction of the N2 binding step is found to have a rate constant of 5.5 ± 0.2 (atm H2)-1 s-1 (7.2 ± 0.3 (mM H2)-1 s-1). Importantly, the reduction of N2 bound to FeMo-cofactor proceeds with a rate constant of 1 ± 0.1 s-1, revealing a previously unrecognized slow step in the Mo-nitrogenase catalytic cycle associated with the chemical transformation of N2 to 2 NH3. Finally, the populations of En states under different reaction conditions are predicted, providing a powerful tool to guide the spectroscopic and mechanistic studies of Mo-nitrogenase.

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation.

Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

An Efficient Viologen-Based Electron Donor to Nitrogenase.

Nitrogenase catalyzes the reduction of N2 to NH3, supporting all biological nitrogen fixation. Electron donors to this enzyme are ferredoxin or flavodoxin (in vivo) and sodium dithionite (in vitro). Features of these electron donors put a limit on spectrophotometric studies and electrocatalytic applications of nitrogenase. Although it is common to use methyl viologen as an electron donor for many low-potential oxidoreductases, decreased nitrogenase activity is observed with an increasing concentration of methyl viologen, limiting its utility under many circumstances. In this work, we suggest that this concentration-dependent decrease in activity can be explained by the formation of a dimer of the radical cation of methyl viologen (Me2V•+)2 at higher methyl viologen concentrations. In addition, viologens functionalized with positively and negatively charged groups were synthesized and studied using spectroscopy and cyclic voltammetry. A sulfonated viologen derivative, 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium radical {[(SPr)2V•]-}, was found to support full nitrogenase activity up to a mediator concentration of 3 mM, while the positively charged viologen derivative was not an efficient reductant of nitrogenase due to the high standard redox potential. The utility of [(SPr)2V•]- as an electron donor for nitrogenase was demonstrated by a simple, sensitive spectrophotometric assay for nitrogenase activity that can provide accurate values for the specific activity and turnover rate constant under argon. Under N2, the formation of ammonia was confirmed. Because of the observed full activity of nitrogenase and low overpotential, [(SPr)2V•]- should also prove to be valuable for nitrogenase electrocatalysis, including bioelectrosynthetic N2 reduction.

Cooperative electrocatalytic alcohol oxidation with electron-proton-transfer mediators.

The electrochemical oxidation of alcohols is a major focus of energy and chemical conversion efforts, with potential applications ranging from fuel cells to biomass utilization and fine-chemical synthesis. Small-molecule electrocatalysts for processes of this type are promising targets for further development, as demonstrated by recent advances in nickel catalysts for electrochemical production and oxidation of hydrogen. Complexes with tethered amines that resemble the active site of hydrogenases have been shown both to catalyse hydrogen production (from protons and electrons) with rates far exceeding those of such enzymes and to mediate reversible electrocatalytic hydrogen production and oxidation with enzyme-like performance. Progress in electrocatalytic alcohol oxidation has been more modest. Nickel complexes similar to those used for hydrogen oxidation have been shown to mediate efficient electrochemical oxidation of benzyl alcohol, with a turnover frequency of 2.1 per second. These compounds exhibit poor reactivity with ethanol and methanol, however. Organic nitroxyls, such as TEMPO (2,2,6,6-tetramethyl-1-piperidine N-oxyl), are the most widely studied electrocatalysts for alcohol oxidation. These catalysts exhibit good activity (1­2 turnovers per second) with a wide range of alcohols and have great promise for electro-organic synthesis. Their use in energy-conversion applications, however, is limited by the high electrode potentials required to generate the reactive oxoammonium species. Here we report (2,2'-bipyridine)Cu/nitroxyl co-catalyst systems for electrochemical alcohol oxidation that proceed with much faster rates, while operating at an electrode potential a half-volt lower than that used for the TEMPO-only process. The (2,2'-bipyridine)Cu(II) and TEMPO redox partners exhibit cooperative reactivity and exploit the low-potential, proton-coupled TEMPO/TEMPOH redox process rather than the high-potential TEMPO/TEMPO+ process. The results show how electron-proton-transfer mediators, such as TEMPO, may be used in combination with first-row transition metals, such as copper, to achieve efficient two-electron electrochemical processes, thereby introducing a new concept for the development of non-precious-metal electrocatalysts.

Dendritic polyglycerol-poly(ethylene glycol)-based polymer networks for biosensing application.

This work describes the formation of a new dendritic polyglycerol-poly(ethylene glycol)-based 3D polymer network as a matrix for immobilization of the redox enzyme periplasmatic aldehyde oxidoreductase to create an electrochemical biosensor. The novel network is built directly on the gold surface, where it simultaneously stabilizes the enzyme for up to 4 days. The prepared biosensors can be used for amperometric detection of benzaldehyde in the range of 0.8-400 µM.

Electrical Wiring of the Aldehyde Oxidoreductase PaoABC with a Polymer Containing Osmium Redox Centers: Biosensors for Benzaldehyde and GABA.

Biosensors for the detection of benzaldehyde and γ-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below -0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A "reagentless" biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10-150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11.

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.