Electron paramagnetic resonance and electron-nuclear double resonance studies of the reactions of cryogenerated hydroperoxoferric-hemoprotein intermediates.

The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein-O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and ß-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and ß-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of ß-chains converts to the S = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis.

Trapping an intermediate of dinitrogen (N2) reduction on nitrogenase.

Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady-state turnover with N2, the S = 3/2 EPR signal (g = [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = 1/2, signal (g = [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at > or = 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A = [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable 1H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor.

Testing if the interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C: ENDOR, ESEEM, and DFT studies of the S = 3/2 resting state in multiple environments.

A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9:homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 35 GHz electron-nuclear double resonance (ENDOR) measurements on 14/15N and 12/13C isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X not equal N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either 14/15N or 13C: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A(14/15NX) < 0.07/0.1 MHz, A(13CX) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = 14/15N/13C/17O, nuclear quadrupole coupling constant e2qQ for X = 14N, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjunction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7" spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 upward arrow:3 downward arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 upward arrow:2 downward arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g2 are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if aiso(14/15NX) < 0.03-0.07/0.05-0.1 MHz and aiso(13CX) < 0.05-0.1 MHz, compared with calculated values of aiso(14/15NX) = 0.3/0.4 MHz and aiso(13CX) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.

Diazene (HN=NH) is a substrate for nitrogenase: insights into the pathway of N2 reduction.

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N2 reduction, is inhibited by H2. This contrasts with the absence of H2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with alpha-195His substituted by Gln and alpha-70Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g1 = 2.09, g2 = 2.01, and g3 approximately 1.98. 15N- and 1H-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N2 by nitrogenase. Implications of these findings for the mechanism of N2 reduction by nitrogenase are discussed.

Electron paramagnetic resonance studies of an integral membrane peptide inserted into aligned phospholipid bilayer nanotube arrays.

This communication reports for the first time the determination of the helical tilt of an integral membrane peptide inserted into aligned phospholipids bilayer nanotube arrays using spin label EPR spectroscopy. Also, we demonstrate herein how the helical tilt of the peptide can be easily calculated using the hyperfine splitting values gleaned from a perpendicularly aligned bilayer in phospholipid bilayer nanotube arrays. EPR spectral simulations were used to verify the method.

Determining the topology of integral membrane peptides using EPR spectroscopy.

This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), was attached to the pore-lining transmembrane domain (M2delta) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14 degrees was calculated for the M2delta peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000-fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 microg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.

Intermediates trapped during nitrogenase reduction of N triple bond N, CH3-N=NH, and H2N-NH2.

A high population intermediate has been trapped on the nitrogenase active site FeMo cofactor during reduction of N2. In addition, intermediates have been trapped during reduction of CH3-N=NH by the alpha-195Gln variant and during reduction of H2N-NH2 by the alpha-70Ala/alpha-195Gln variant. Each of these trapped states shows an EPR signal arising from an S = 1/2 state of the FeMo cofactor. 15N ENDOR shows that each intermediate has a nitrogenous species bound to the FeMo cofactor, with a single type of N seen for each bound intermediate. The g tensors are unique to each intermediate, g(e) = [2.084, 1.993, 1.969], g(m) = [2.083, 2.021, 1.993], g(l) = [2.082, 2.015, 1.987], as are the 15N hyperfine couplings at g1, which suggests that three distinct stages of NN reduction may have been trapped. The 1H ENDOR spectra show that the N2 intermediate is at a distinct and earlier stage of reduction from the other two, so at least two stages of NN reduction have been trapped. Some possible structures of the hydrazine intermediate are presented.

The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM evidence that it is not a nitrogen.

X-ray crystallographic study of the nitrogenase MoFe protein revealed electron density from an atom (denoted X) inside the active-site metal cluster, the [MoFe7S9:homocitrate] FeMo-cofactor. The electron density associated with X is consistent with a single N, O, or C atom. We now have tested whether X is an N or not by comparing the Q-band ENDOR and ESEEM signals from resting-state (S = 3/2) MoFe protein and NMF-extracted FeMo-co from bacteria grown with either 14N or 15N as the exclusive N source. All of the 14N or 15N signals associated with the protein are lost upon extraction of the FeMo-co. We interpret this as strong evidence that X is not an N.

Trapping a hydrazine reduction intermediate on the nitrogenase active site.

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.

Trapping H- bound to the nitrogenase FeMo-cofactor active site during H2 evolution: characterization by ENDOR spectroscopy.

We here show that the iron-molybdenum (FeMo)-cofactor of the nitrogenase alpha-70(Ile) molybdenum-iron (MoFe) protein variant accumulates a novel S = (1)/(2) state that can be trapped during the reduction of protons to H(2). (1,2)H-ENDOR measurements disclose the presence of two protons/hydrides (H(+/)(-)) whose hyperfine tensors have been determined from two-dimensional field-frequency (1)H ENDOR plots. The two H(+/)(-) have large isotropic hyperfine couplings, A(iso)( )() approximately 23 MHz, which shows they are bound to the cofactor. The favored analysis for these plots indicates that the two H(+/)(-) have the same principal values, which indicates that they are chemically equivalent. The tensors are further related to each other by a permutation of the tensor components, which indicates an underlying symmetry of binding relative to the cofactor. At present, no model for the structure of the iron-molybdenum (FeMo)-cofactor in the S = (1)/(2) state trapped during the reduction of H(+) can be shown unequivocally to satisfy all of the constraints generated by the ENDOR analysis. The data disfavors any model that involves protonation of sulfides, and thus suggests that the intermediate instead contains two chemically equivalent bound hydrides; it appears unlikely that these are terminal monohydrides.

An organometallic intermediate during alkyne reduction by nitrogenase.

Nitrogenase is the metalloenzyme that catalyzes the nucleotide-dependent reduction of N(2), as well as reduction of a variety of other triply bonded substrates, including the alkyne, acetylene. Substitution of the alpha-70(Val) residue in the nitrogenase MoFe protein by alanine expands the range of substrates to include short-chain alkynes not reduced by the unaltered protein. Rapid freezing of the alpha-70(Ala) nitrogenase MoFe protein during reduction of the alkyne propargyl alcohol (HC triple bond CH(2)OH; PA) traps an S = (1)/(2) intermediate state of the active-site metal cluster, the FeMo-cofactor. We have combined CW and pulsed (13)C ENDOR (electron-nuclear double resonance) with two quantitative 35 GHz (1,2)H ENDOR techniques, Mims pulsed ENDOR and the newly devised "stochastic field-modulated" ENDOR, to study this intermediate prepared with isotopically substituted ((13)C, (1,2)H) propargyl alcohol in H(2)O and D(2)O buffers. These measurements allow the first description of a trapped nitrogenase reduction intermediate. The S = (1)/(2) turnover intermediate generated during the reduction of PA contains the 3-carbon chain of PA and exhibits resolved (1,2)H ENDOR signals from three protons, two strongly coupled (H(a)) and one weakly coupled (H(b)); H(a)(c) originates as the C3 proton of PA, while H(a)(s) and H(b) are solvent-derived. The two H(a) protons have identical hyperfine tensors, despite having different origins. The equality of the (H(a)(s), H(a)(c)) hyperfine tensors strongly constrains proposals for the structure of the cluster-bound reduced PA. Through consideration of model structures found in the Cambridge Structural Database, we propose that the intermediate contains a novel bio-organometallic complex in which a reduction product of propargyl alcohol binds as a metalla-cyclopropane ring to a single Fe atom of the Fe-S face of the FeMo-cofactor that is composed of Fe atoms 2, 3, 6, and 7. Of the two most attractive structures, one singly reduced at C3 (4), the other being the doubly reduced allyl alcohol product (6), we tentatively favor 6 because of the "natural" assignment it affords for H(b).

Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: trapping propargyl alcohol with an alpha-70-substituted MoFe protein.

Substitution of the MoFe protein alpha-70(Val) residue with Ala or Gly expands the substrate range of nitrogenase, allowing the reduction of larger alkynes, including propargyl alcohol (HC[triple bond]CCH(2)OH). Herein, we report characterization of the alpha-70(Val)(-->)(Ala) MoFe protein with propargyl alcohol trapped at the active site. The alpha-70(Ala) variant MoFe protein was rapidly frozen during reduction of propargyl alcohol, resulting in the conversion of the resting-state FeMo-cofactor EPR signal (S = 3/2 and g = [4.41, 3.60, 2.00]) to a new state (S = 1/2 and g = [2.123, 1.998, 1.986]). This EPR signal of the new state increased in intensity with increasing propargyl alcohol concentration, consistent with the binding of a single substrate. The EPR signal of the propargyl alcohol state showed temperature and microwave power dependencies markedly different from those of the classic FeMo-cofactor EPR signal, consistent with the difference in spin. The new state is analogous to that induced by the binding of the inhibitor CO ("lo CO" state) to FeMo-cofactor in the wild-type MoFe protein. The (13)C ENDOR spectrum of the alpha-70(Ala) MoFe protein with trapped (13)C-labeled propargyl alcohol exhibited three well-resolved (13)C doublets centered at the (13)C Larmor frequency with isotropic hyperfine couplings of approximately 3.2, approximately 1.4, and approximately 0.7 MHz, indicating that the alcohol (or a fragment) is coordinated to the cofactor. The results presented here localize the binding site of propargyl alcohol to one [4Fe-4S] face of FeMo-cofactor and indicate roles for the alpha-70(Val) residue in controlling FeMo-cofactor reactivity.

The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM show it is not an exchangeable nitrogen.

A recent high-resolution X-ray crystallographic study (1.16 A) of the Azotobacter vinelandii nitrogenase MoFe protein revealed a previously undetected electron density associated with the active site FeMo-cofactor. The density is located inside the cluster at the center of the "trigonal prism" of six irons and is assigned to a species "X". The identity of species X was not resolved, although the electron density is consistent with a single N, O, or C atom. One proposal is that X is an N atom that derives from and exchanges with N from N2 during catalysis. In the present study, we have examined this possibility by employing 14N and 15N isotopes of N2 along with ENDOR and ESEEM spectroscopies. The WT MoFe protein and alpha-359Arg-->Lys and alpha-381Phe-->Leu variants were allowed to turn over in the presence of 14N2 or 15N2, and then were examined as resting enzymes by ENDOR and ESEEM at X- and Q-bands to look for all 14N and 15N signals coupled to the electron spin of the FeMo-cofactor and to determine if any exchanged during turnover. We have found five peaks in Q-band pulsed ENDOR spectra that appear to arise not only from previously reported N1/N2, which give rise to the ESEEM, but also from one or two additional coupled nitrogens. None of the ENDOR and ESEEM signals vanish or are altered by catalytic turnover with 15N2, and no new 15N signal is detected, leading to the conclusion that if species X is a nitrogen atom, it does not exchange during dinitrogen reduction.