Electron Transfer Precedes ATP Hydrolysis during Nitrogenase Catalysis

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein

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

Phototrophic N2 and CO2 Fixation Using a Rhodopseudomonas palustris-H2 Mediated Electrochemical System With Infrared Photons

A promising approach for the synthesis of high value reduced compounds is to couple bacteria to the cathode of an electrochemical cell, with delivery of electrons from the electrode driving reductive biosynthesis in the bacteria. Such systems have been used to reduce CO2 to acetate and other C-based compounds. Here, we report an electrosynthetic system that couples a diazotrophic, photoautotrophic bacterium, Rhodopseudomonas palustris TIE-1, to the cathode of an electrochemical cell through the mediator H2 that allows reductive capture of both CO2 and N2 with all of the energy coming from the electrode and infrared (IR) photons. R. palustris TIE-1 was shown to utilize a narrow band of IR radiation centered around 850 nm to support growth under both photoheterotrophic, non-diazotrophic and photoautotrophic, diazotrophic conditions with growth rates similar to those achieved using broad spectrum incandescent light. The bacteria were also successfully cultured in the cathodic compartment of an electrochemical cell with the sole source of electrons coming from electrochemically generated H2, supporting reduction of both CO2 and N2 using 850 nm photons as an energy source. Growth rates were similar to non-electrochemical conditions, revealing that the electrochemical system can fully support bacterial growth. Faradaic efficiencies for N2 and CO2 reduction were 8.5 and 47%, respectively. These results demonstrate that a microbial-electrode hybrid system can be used to achieve reduction and capture of both CO2 and N2 using low energy IR radiation and electrons provided by an electrode

Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves

Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle

Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation to release of Pi. Because the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein–protein interactions during the Fe protein cycle. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc

Evidence That the P\u3csub\u3ei\u3c/sub\u3e Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein–protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle

Optimizing Nitrogen Fixation and Recycling for Food Production in Regenerative Life Support Systems

Nitrogen (N) recycling is essential for efficient food production in regenerative life support systems. Crew members with a high workload need 90–100 g of protein per person per day, which is about 14 g of N, or 1 mole of N, per person per day. Most of this N is excreted through urine with 85% as urea. Plants take up N predominantly as nitrate and ammonium, but direct uptake as urea is possible in small amounts. Efficient N recycling requires maintenance of pH of waste streams below about 7 to minimize the volatilization of N to ammonia. In aerobic reactors, continuous aerobic conditions are needed to minimize production and volatilization of nitrous oxide. N is not well recycled on Earth. The energy intensive Haber–Bosh process supplies most of the N for crop production in terrestrial agriculture. Bacterial fixation of dinitrogen to ammonium is also energy intensive. Recycling of N from plant and human waste streams is necessary to minimize the need for N fixation. Here we review approaches and potential for N fixation and recycling in regenerative life support systems. Initial estimates indicate that nearly all the N from human and plant waste streams can be recovered in forms usable for plants

Kinetic and Spectroscopic Analysis of the Inactivating Effects of Nitric Oxide on the Individual Components of Azotobacter vinelandii Nitrogenase

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 2.03) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Avl) were more complex than for Av2. A time-dependent inactivation of Avl activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 P a ) . In addition, the effects of NO on Avl were significantly affected by the presence of sodium dithionite. In fact, kinetic evidence suggests that an Avl-catalyzed, NO-dependent consumption of dithionite occurs before Avl is inactivated by NO. A correlation between UV-visible and EPR spectral features and the extent of NO inactivation has been established. The inactivation of either nitrogenase component by NO did not lead to aggregation or dissolution into their constitutive subunits. However, NO inactivation did cause changes in both proteins since neither NO-treated protein inhibited C2H2-reducing activity in assays containing equimolar concentrations of untreated protein. The effects of NO on both nitrogenase components are interpreted in terms of the known reactivity of NO with Fe-S centers

Sequential and Differential Interaction of Assembly Factors During Nitrogenase MoFe Protein Maturation

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states

Nitrogenase bioelectrocatalysis: heterogeneous ammonia and hydrogen production by MoFe protein

notrogenase is the only enzyme known to catalyze the reduction of N2 to 2NH3. In vivo, the MoFe protein component of nitrogenase is exclusively reduced by the ATP-hydrolyzing Fe protein in a series of transient association/dissociation steps that are linked to the hyderolysis of two ATP for each electron transeferred. We report MoFe protein immobilized at an electrode surface, where cobaltocene (as an electron mediator that can be observed in real time at a carbon electrode) is used to reduce the MoFe protein (independent of the Fe protein and of ATP hydrolysis) and support the bioelectrocatalytic reduction of protons to dihydrogen, azide to ammonia, and nitrit to ammonia. Bulk bioelectrosynthetic N3 or NO2 reduction (50 mM) for 30 minutes yielded 70 +- 9 nmol NH3 and 234 +- 62 nmol NH3, with NO2 reduction operating at high faradaic efficiency