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

• Published in: Inorganic chemistry 2007-12, Vol.46 (26), p.11437-11449
• Main Authors: Lukoyanov, Dmitriy, Pelmenschikov, Vladimir, Maeser, Nathan, Laryukhin, Mikhail, Yang, Tran Chin, Noodleman, Louis, Dean, Dennis R, Case, David A, Seefeldt, Lance C, Hoffman, Brian M
• Format: Article
• Language: English
• Subjects: Carbon - chemistry; Iron - chemistry; Iron - metabolism; Models, Molecular; Molecular Conformation; Molybdenum - chemistry; Molybdenum - metabolism; Nitrogen - chemistry; Nitrogenase - metabolism; Sulfur - chemistry
• ISSN: 0020-1669; 1520-510X
• DOI: 10.1021/ic7018814

A high-resolution (1.16 Å) 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 ≠ 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(14/15N X ) < 0.07/0.1 MHz, A(13C X ) < 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 e 2 qQ 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↑:3↓) iron atoms of the [6Fe] prismane “waist” of FeMo-co; this would rule out the “BS6” assignment (4↑:2↓ for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g 2 are sufficiently large that they should have been detected:  we suggest that the experimental results are compatible with X = N only if a iso(14/15N X ) < 0.03−0.07/0.05−0.1 MHz and a iso(13C X ) < 0.05−0.1 MHz, compared with calculated values of a iso(14/15N X ) = 0.3/0.4 MHz and a iso(13C X ) = 1 MHz. However, the DFT uncertainties are large enough