Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N 2 for H 2by Dmitriy Lukoyanov, Zhi-Yong Yang, Nimesh Khadka, Dennis R. Dean, Lance C. Seefeldt, Brian M. Hoffman

J. Am. Chem. Soc.


Chemistry (all) / Colloid and Surface Chemistry / Biochemistry / Catalysis


Identification of a Key Catalytic Intermediate Demonstrates That

Nitrogenase Is Activated by the Reversible Exchange of N2 for H2

Dmitriy Lukoyanov,‡,∥ Zhi-Yong Yang,†,∥ Nimesh Khadka,† Dennis R. Dean,*,§ Lance C. Seefeldt,*,† and Brian M. Hoffman*,‡ ‡Departments of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States †Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States §Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States *S Supporting Information

ABSTRACT: Freeze-quenching nitrogenase during turnover with N2 traps an S = 1/2 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. ■ INTRODUCTION

Biological nitrogen fixationthe reduction of N2 to two NH3 moleculesis primarily catalyzed by the Mo-dependent nitrogenase. This enzyme comprises an electron-delivery Fe protein and MoFe protein, a dimer of dimers that contains two copies of the active-site FeMo-cofactor (FeMo-co).1,2 A suggested limiting stoichiometry for nitrogen fixation,3 + + + → + + + − +N 8e 16ATP 8H 2NH H 16ADP 16P 2 3 2 i (1) incorporates an obligatory formation of 1 mol of H2 per mole of N2 reduced, and thus a puzzling requirement for two reducing equivalents and four ATP beyond the chemical requirement for N2 reduction. 1,2 This stoichiometry is embodied in a kinetic framework for nitrogenase function provided by the Lowe−Thorneley (LT) model,1,2,4 which describes transformations among catalytic intermediates denoted En, where n is the number of electrons and protons (n = 0−8) delivered to one-half of the MoFe protein (Figure 1A). In this model, N2 reduction requires activation of the

MoFe protein to the pivotal E4(4H) state (see the kinetic scheme of Figure 1, where the legend explains notation), in which ENDOR has shown FeMo-co to have accumulated four reducing equivalents stored in the form of two [Fe−H−Fe] bridging hydrides,5−7 presumably with two protons bound to sulfides of FeMo-co (Figure 1B, left).5,7−9 We recently proposed8,10 and subsequently provided experimental evidence11 that nitrogenase is activated for N2 binding and reduction through reductive elimination (re)12−15 of the two bridging hydrides of E4(4H) to form H2 (Figure 1B), thereby experimentally supporting the stoichiometry of eq 1.

As part of the development of the re mechanism we used advanced paramagnetic techniques to characterize two nitrogenous En intermediates that are associated with states in the

LT scheme subsequent to N2 binding, n ≥ 4 (Figure 1), and that are common to turnover of remodeled nitrogenase with

N2H2, Me-N2H, N2H4, NO2 −, and H2NOH. One is a nonKramers (S ≥ 2) state, denoted H, that is assigned as E7, with bound [-NH2]; the second is a Kramers (S = 1/2) state, denoted I, assigned as E8, with bound NH3. 8,10,16 During turnover of wild-type nitrogenase with N2 an additional intermediate state with S = 1/2 was trapped, but not identified.17−19 With a half-integer spin, like the E0 resting state, this state must differ from E0 by the accumulation of an n = even number of [e−/H+] to FeMo-co. This state, herein denoted as EG, must further correspond to an En state formed subsequent to binding N2, n = 4, 6, or 8, because it gives 15N

ENDOR signals when trapped using 15N2 substrate. The previous assignment of I as E8, a product (NH3)-bound state,8,10 implies an assignment of EG to E4 or E6. The notation,

Received: January 5, 2015

Published: March 5, 2015

Article © 2015 American Chemical Society 3610 DOI: 10.1021/jacs.5b00103

J. Am. Chem. Soc. 2015, 137, 3610−3615

EG, in fact was adopted because if the states of Figure 1A were to be labeled sequentially by letters beginning with E0 = A, then

E4 would be E and E6 would be G.

Because of the relatively low accumulation of EG in freezequenched samples, to date neither ENDOR measurements nor the use HYSCORE, as successfully applied to intermediate I,20 have yielded an assignment of EG. To identify EG, we therefore here turn to a quench-cryoannealing relaxation protocol developed to determine the reduction level, n, of an EPRactive freeze-trapped En intermediate state. 6 The trapped state is allowed to relax to the resting E0 state in frozen medium, at temperatures T ≤ −20 °C, well below the melting temperature of the buffered samples, T ≈ 0 °C. Keeping the sample frozen prevents any additional accumulation of reducing equivalents, because binding of reduced Fe protein to and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. As recently confirmed,21 the frozen intermediate can relax toward the resting state only through steps that release a stable species from FeMo-co. The En states formed prior to N2 binding lose 2 equiv per relaxation step by releasing