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Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects † ⊥ † ‡ § Nimesh Khadka, Ross D

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Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects † ⊥ † ‡ § Nimesh Khadka, Ross D Article pubs.acs.org/JACS Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects † ⊥ † ‡ § Nimesh Khadka, Ross D. Milton, Sudipta Shaw, Dmitriy Lukoyanov, Dennis R. Dean, ⊥ ¶ ‡ † Shelley D. Minteer, Simone Raugei, Brian M. Hoffman,*, and Lance C. Seefeldt*, † Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States ‡ Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States § Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States ⊥ Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States ¶ Pacific Northwest National Laboratory, Richland, Washington 99352, United States *S Supporting Information ABSTRACT: Nitrogenase catalyzes the reduction of dini- trogen (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 com- peting 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 reduc- tion 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 H2OandD2O, 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 ff Fe-hydride, and explains the observed H/D isotope e ect. 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. See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. 9 Downloaded via PACIFIC NORTHWEST NATL LABORATORY on December 27, 2018 at 23:43:12 (UTC). ■ INTRODUCTION electrons/protons delivered. Nitrogenase is activated for N2 Bacterial nitrogenase catalyzes the reduction of dinitrogen (N ) reduction via re of H2 after the accumulations of four electrons/ 2 protons, stored as two [Fe−H−Fe] bridging hydrides and two to two molecules of ammonia (NH3), making the largest con- 10,11 fi protons (E4(4H) state). The re mechanism is kinetically tribution of xed nitrogen in the global biogeochemical 4,7 1 and thermodynamically reversible, and the hydrides in the Ncycle. The Mo-dependent nitrogenase is composed of an Fe 5,6 protein that delivers electrons to the MoFe protein, where the E4(4H) state are photolytically active. In a parallel, compet- “ ” active site FeMo-cofactor (7Fe-9S-1Mo-1C-1R homocitrate) is itive reaction, nitrogenase functions as a hydrogenase , pro- ducing H through the protonation of a metal-hydride and bound. Recent work has established that the reduction of N2 at 2 the active site FeMo-cofactor involves reductive elimination relaxation to a two-electron-less reduced En‑2 state (Figure 1B). − − The delivery of electrons to the MoFe protein and the active (re) of two bridging Fe H Fe hydrides to form H2 in a reac- tion that is linked to N binding and activation to a metal- site FeMo-cofactor occurs one electron at a time during the 2 − bound diazene level intermediate (Figure 1).2 8 The first four transient association of the Fe protein component of nitrogenase electrons and protons are accumulated on the FeMo-cofactor stepwise and are stored as Fe-bound hydrides in states desig- Received: July 13, 2017 nated as En where the subscript n indicates the number of Published: August 29, 2017 © 2017 American Chemical Society 13518 DOI: 10.1021/jacs.7b07311 J. Am. Chem. Soc. 2017, 139, 13518−13524 Journal of the American Chemical Society Article reference and glassy carbon working electrodes were purchased from CH Instruments, Inc. Bacterial Growth and Protein Purification. Azotobacter vinelandii strains DJ995 (wild-type MoFe protein), DJ1003 (apo- MoFe protein), DJ 1316 (α-70Val→Ala/α-195His→Gln), and DJ1373 (α-70Val→Ile) were grown, and seven histidine-tagged proteins were purified as previously reported. Strain DJ939 (β-98Tyr→His) was also grown and the corresponding non-His tagged MoFe protein was purified as described.17 nifX protein was purified and complexed with N-methyl formamide (NMF) isolated FeMo-cofactor using a protocol as described previously.18 Bioelectrocatalysis. All experiments were conducted in an Ar-filled glovebox. Purified MoFe protein (20 mg mL−1,15μL) was mixed with polyvinylamine (15 μLat10mgmL−1) and EGDGE (2 μL at 10% v/v). This mixture (5 μL) was applied to the surface of a planar glassy carbon disk electrode (3-mm diameter) and dried under reduced humidity for 1 h. Cyclic voltammetry was carried out under an Ar atmosphere in 250 mM HEPES buffer (pH meter reading of 7.2 μ Figure 1. (A) Schematic representation of the FeMo-cofactor with for H2O and 6.8 for D2O) using 667 M cobaltocene/cobaltocenium 0 − α-Cys275, α-His442, and R-homocitrate as ligands. The red highlighted (bis-cyclopentadienyl cobalt (III/II); E = 1.25 V vs SCE) (abbre- square represents the catalytically active 4Fe−4S face of the FeMo- viated CC) as the electron mediator, Pt as the counter electrode, and cofactor. (B) E states of FeMo-cofactor during accumulation of the a saturated calomel electrode (SCE) as the reference electrode. n fl first four electrons/protons, along with the reductive elimination/ Bis(cyclopentadienyl)cobalt(III) hexa uorophosphate was used to “ ” prepare a 6 mM stock in 250 mM HEPES H O and 250 mM HEPES oxidative addition (re/oa) mechanism at E4(4H). The 2N2H inter- 2 mediate implies a species at the diazene reduction level of unknown D2O. The pH of both mixtures were adjusted to pH 7.2 and pD 7.2, structure and coordination geometry. respectively. The calculated volume was then added from these stocks to 250 mM HEPES H2O pH 7.2 and 250 mM HEPES D2O pD 7.2 to 12 achieve the final concentration of 667 μM cobaltocenium. Solvent with the MoFe protein component. The Fe protein delivers ff one electron from its [4Fe−4S] center in a process coupled to deuterium kinetic isotope e ect experiments were undertaken as a function of solvent composition (proton inventory) by mixing solu- the hydrolysis of 2ATP to 2ADP/Pi. The electron is delivered tions containing the buffer made in D OorHO. The current mea- to the MoFe protein active-site FeMo-cofactor, with the 2 2 13 sured with a mole fraction of D2O in the mixture (n), denoted (in), was P-cluster acting as an electron carrier intermediary. Earlier divided by the current in 100% H2O(in/i0), and was plotted against fi fi work conducted with the arti cial electron donor sodium dithio- the mole fraction of D2O(n), and the data were well- tted to the nite indicated that the dissociation of the oxidized Fe protein Gross-Butler equation for a one-proton transfer reaction19 with 2 bound ADP was rate-limiting for the overall nitrogenase ⎛ ⎞ 14 in 1 catalysis. Recent studies using the natural electron donor, =−11⎝⎜⎟ − ⎠n flavodoxin, have revealed that the overall rate-limiting step is i0 KIE associated with the release of Pi before the fast dissociation of =−1 an (1) the oxidized Fe protein with 2ADP from the MoFe protein.15 KIE− 1 As the rate-limiting step for nitrogenase catalysis is associated a = with electron delivery by the Fe protein, it is not possible to KIE kinetically probe the substrate reduction chemistry at FeMo- ff The noncatalytic current was determined when bovine serum cofactor, and in particular measurements of isotope e ects on albumin replaced the nitrogenase MoFe-protein. The noncatalytic current the rates of product formation during enzymatic turnover. wasmeasuredateachratioofH2OandD2O. In all cases, the non- Recently, we demonstrated that it is possible to deliver elec- catalytic current was very low compared to the catalytic current and did trons to the MoFe protein without the Fe protein through a not show any kinetic isotope effect (KIE). The noncatalytic current was mediated electrochemical approach.16 It was demonstrated that subtracted from the observed current at each mole fraction to get the net electrons could be delivered to electrode-confined MoFe pro- catalytic current that was used for the proton inventory studies. Quantum Chemical Calculations. A quantum chemical analysis tein without the Fe protein, and thus without ATP hydrolysis, ff of the e ect of the H/D on the formation of H2 was performed on a and that this can drive the reduction of several substrates, as fi α Cys 16 simpli ed model that comprises the FeMo-co and ligands -275 , well as the protonation of a metal-hydride to make H2. This α-442His (Azotobacter vinelandii numbering), and R-homocitrate, which approach thus creates a new rate-limiting step not associated were modeled as methylthiolate, imidazole, and dimethyl glycolate, with events in the Fe protein, offering the possibility to probe respectively.
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