Testing if the Interstitial Atom,X, of the Nitrogenase Molybdenum−Iron Cofactor Is N or C:  ENDOR, ESEEM, and DFT Studies of theS=³/₂Resting State in Multiple Environments

Abstract

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.

QM/MM Study of the Nitrogenase MoFe Protein Resting State: Broken-Symmetry States, Protonation States, and QM Region Convergence in the FeMoco Active Site

“…All of our QM/MM calculations have used broken-symmetry SCF solutions (with M S =3/2) using the broken-symmetry solution known as BS7 as proposed by Noodleman 11 . This broken-symmetry solution has been found by many research groups 11,18,20,21,7,28 to be the lowest energy one which can be explained as being mainly due to presence of more favorable antiferromagnetic interactions between Fe atoms than other BS solutions.…”

“…Noodleman and coworkers mainly used small models in their pioneering studies 8,9,10,11 but discussed the effect of the protein environment in a later study 12 . Studies by Norskov 13,14,15,16 , Blöchl 17,18 , Kästner 19 , Szilagyi 20 , Dance 21,22 and McKee 23 have used minimal cofactor models.…”

Iron–dinitrogen coordination chemistry: Dinitrogen activation and reactivity

“…1) [19]. The identity of this central atom is still hotly debated, but from the electron density data it is expected to be either C, N, or O [20][21][22][23][24][25][26][27][28][29].…”

PhB( tBuIm) 3 ? : Synthesis, Structure, and Reactivity of an Iron(V) Nitride

“…While iron bound ''surface nitride'' species are observed on the catalyst surface in the Haber-Bosch process [9], recent X-ray diffraction studies on nitrogenase suggest an interstitial atom in the center of the iron-sulfur cluster of the FeMo cofactor [10]. Although it is tempting to suggest a nitride anion at the site of biological nitrogen reduction, the nature of this atom is controversial and still under debate [11,12]. Accordingly, the synthesis and characterization of model complexes is critical to delineating the reactivity of iron nitrides and their possible role in ammonia synthesis.…”