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Unification of Reaction Pathway and Kinetic Scheme for N2 Reduction Catalyzed by Nitrogenase Dmitriy Lukoyanova, Zhi-Yong Yangb, Brett M

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Unification of Reaction Pathway and Kinetic Scheme for N2 Reduction Catalyzed by Nitrogenase Dmitriy Lukoyanova, Zhi-Yong Yangb, Brett M Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase Dmitriy Lukoyanova, Zhi-Yong Yangb, Brett M. Barneyb,1, Dennis R. Deanc, Lance C. Seefeldtb, and Brian M. Hoffmana,2 aDepartment of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208; bDepartment of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322; and cDepartment of Biochemistry, Virginia Tech, 110 Fralin Hall, Blacksburg, VA 24061 Contributed by Brian M. Hoffman, February 7, 2012 (sent for review January 10, 2012) Nitrogenase catalyzes the reduction of N2 and protons to yield two at the fifth hydrogenation step (8). However, this conclusion left NH3 and one H2. Substrate binding occurs at a complex organo- many questions unresolved, in particular the correspondence be- metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle tween intermediates formed along the reaction pathway and the involves the sequential delivery of eight electrons/protons to this LT kinetic scheme for the accumulation of eight reducing equiva- cluster, and this process has been framed within a kinetic scheme lents during catalysis. developed by Lowe and Thorneley. Rapid freezing of a modified Intermediates formed during turnover have been trapped by nitrogenase under turnover conditions using diazene, methyldia- freeze-quench methods and studied by paramagnetic resonance ¼ zene (HN N-CH3), or hydrazine as substrate recently was shown techniques. The resting-state FeMo-co of E0 exhibits an EPR- ¼ 1 I ¼ 3 to trap a common S 2 intermediate, designated . It was further active, S 2 spin state. When nitrogenase is freeze-quenched concluded that the two N-atoms of N2 are hydrogenated alter- during turnover, the resting-state EPR spectrum partially or fully nately (“Alternating” (A) pathway). In the present work, Q-band disappears as FeMo-cofactor accumulates electrons. Accumula- 95 ¼ CW EPR and Mo ESEEM spectroscopy reveal such samples also tion of an even number of electrons generates En, n even, in- contain a common intermediate with FeMo-co in an integer-spin termediates with EPR-active, odd-electron (half-integer spin; state having a ground-state “non-Kramers” doublet. This species, 1 3 Kramers) FeMo-co states (S ¼ 2 ; 2) (3, 4). Such states have been designated H, has been characterized by ESEEM spectroscopy using , , observed upon freeze-quench of wild-type and amino-acid substi- 14 15 1 2 CHEMISTRY a combination of N isotopologs plus H isotopologs of tuted MoFe proteins during turnover with a variety of different H methyldiazene. It is concluded that: has NH2 bound to FeMo-co substrates (3, 4). However, the signals from Kramers forms of and corresponds to the penultimate intermediate of N2 hydroge- FeMo-co in the quenched samples never quantitate to the total nation, the state formed after the accumulation of seven electrons/ FeMo-co present, indicating that apparently EPR-silent states of I protons and the release of the first NH3; corresponds to the final FeMo-co must also exist. These silent MoFe protein states con- intermediate in N2 reduction, the state formed after accumulation tain FeMo-co with an even number of electrons, and correspond of eight electrons/protons, with NH still bound to FeMo-co prior to ¼ ¼ 2 þ 1 ¼ 0–3 3 to En, n odd (n m , m ) intermediates in the LT release and regeneration of resting-state FeMo-co. A proposed uni- scheme. These quenched samples may include states with dia- fication of the Lowe-Thorneley kinetic model with the “prompt” magnetic FeMo-co, but Mössbauer studies indicated the presence BIOCHEMISTRY alternating reaction pathway represents a draft mechanism for of reduced FeMo-co in integer-spin (S ¼ 1; 2, .....), “non-Kra- N2 reduction by nitrogenase. mers (NK)” states (9). Although NK-EPR signals at conventional microwave frequencies are well known for other enzymes (10, EPR ∣ ESEEM ∣ non-Kramers 11), until now, no EPR signal from an integer-spin form of FeMo-co has been detected. itrogen fixation—the reduction of N2 to two NH3 molecules Recently, rapid freezing during turnover of a doubly-substi- N—is catalyzed by the enzyme nitrogenase according to the tuted nitrogenase MoFe protein (α-70Val→Ala, α-195His→Gln), limiting stoichiometry (1, 2): which favors reduction of large nitrogenous substrates (3, 4), with diazene, methyldiazene (HN¼N-CH3), or hydrazine as substrate − þ N2 þ 8e þ 8H þ 16MgATP → 2NH3 þ H2 þ 16MgADP 1 was shown to trap a common S ¼ 2 intermediate (denoted I) (8). 95 þ 16Pi [1] Here, Q-band CW EPR and Mo ESEEM (electron spin-echo envelope modulation) spectroscopy reveal that these samples H The Mo-dependent enzyme studied here (2–4) consists of two also contain a second common intermediate, denoted , one component proteins, denoted the Fe protein and the MoFe pro- that is observed in which FeMo-co is in an EPR-active integer- spin state with a ground-state NK doublet. The NK-EPR signal tein. The former delivers electrons one-at-a-time to the MoFe 95 protein, where they are utilized at the active site iron-molybde- in samples prepared with Mo-enriched FeMo-co and with 14 15 num cofactor ([7Fe-9S-Mo-C-R-homocitrate]; FeMo-co) to re- ; N-enriched substrates has been studied by pulsed-EPR mea- duce substrate (2, 3). In the Lowe-Thorneley (LT) kinetic surements of the nuclear modulation of the electron spin-echo scheme for N2 reduction by nitrogenase (2, 5, 6), the eight steps (ESE) amplitude, denoted NK-ESEEM (12). These measure- 1 – H of electron/proton delivery implied by Eq. are denoted En, ments allow us to infer the state of the N N bond in and where n ¼ 0 to 8, with E0 representing the resting-state enzyme. the relationship of H to intermediate, I. Most importantly, the It is known that N2 binds only after the MoFe protein accumu- analysis allows us to assign the correspondence of both intermedi- lates three or four electrons (E3 or E4 states), with N2 reduction ates with specific En states, and to infer how the hydrogenated then proceeding along a reaction pathway that comprises the se- quence of intermediate states generated as N2 bound to FeMo-co −∕ þ Author contributions: D.L., Z.-Y.Y., B.M.B., D.R.D., L.C.S., and B.M.H. designed research; undergoes six steps of hydrogenation (e H delivery to sub- D.L. performed research; Z.-Y.Y. and B.M.B. contributed new reagents/analytic tools; strate) (1–4, 7). Recently, we concluded that nitrogenase follows D.L. analyzed data; and D.L., Z.-Y.Y., B.M.B., D.R.D., L.C.S., and B.M.H. wrote the paper. an “alternating” (A) reaction pathway, in which the two N’s are The authors declare no conflict of interest. hydrogenated alternately, with a hydrazine-bound intermediate 1Present address: Department of Bioproducts and Biosystems Engineering, University of formed after four steps of hydrogenation, and with cleavage of Minnesota, St. Paul, MN 55108. the hydrazine N-N bond and liberation of the first NH3 only 2To whom correspondence should be addressed. E-mail:[email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1202197109 PNAS ∣ April 10, 2012 ∣ vol. 109 ∣ no. 15 ∣ 5583–5587 Downloaded by guest on October 2, 2021 reaction intermediates, diazene and hydrazine, join the N2 reduc- of a field establishes the coupling and introduces modulation, tion pathway. These conclusions integrate the sequence of N2 with a depth that increases quadratically with B (15). reduction intermediates (the reaction “pathway”) with the LT ki- As illustrated in Fig. 2A, the NK-ESEEM time-waves for the netic scheme for the accumulation of the eight reducing equiva- NK intermediates trapped during turnover with the corresponding 14 15 lents (and protons), thereby generating a draft mechanism for Nand N isotopologs of N2H2,N2H4,andHN2CH3 substrates nitrogen fixation by nitrogenase. are identical at all fields, indicating that a common intermediate, denoted H, is trapped during turnover with all three substrates. Results Fig. 2A, Left and Fig. 2C, Left further show that 95Mo enrichment → The EPR spectra of the doubly substituted α-70Val Ala, of α-70Val→Ala, α-195His→Gln MoFe protein produces significant → α-195His Gln MoFe protein trapped during turnover in the pre- change of the NK-ESEEM time-wave. This establishes that the sence of hydrazine, diazene, and methyldiazene show a significant NK-EPR signal of H arises from the Mo-containing FeMo-co ¼ 3 loss of the S 2 resting state of FeMo-co and the appearance of a in an integer-spin state, and not the all-iron electron-transfer- 1 S ¼ 2 signal from intermediate I in the vicinity of g ¼ 2, along active P cluster also present in the MoFe protein, or even the 1þ with the spectrum from the ½4Fe4S cluster of the Fe protein [4Fe-4S] cluster of the Fe protein. The absence of any signal in 1 14 15 (Fig. 1). Recent H, ; N ENDOR and HYSCORE spectro- Fig. 1 from the P cluster further indicates that all electrons deliv- scopic experiments demonstrated that I represents a late stage ered to MoFe protein of H during turnover reside on FeMo-co. of nitrogen fixation, when the first ammonia molecule already The “turn-on” of nuclear modulation for H with applied field is ¼ 2 has been released (8), and only a [NHx](x or 3) fragment quite gradual in comparison with that observed for the carboxy- of substrate is bound to FeMo-co (8). late-bridged diiron centers (16). In addition to the hyperfine cou- We here note that samples freeze-trapped during turnover pling and g-value along the unique magnetic axis of the center, with all three substrates also show an additional broad EPR signal Ajj and gjj, there are three factors that influence the field depen- at low field in Q-band spectra collected at a temperature of 2 K dence of the modulation depth (15).
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