Dataset Information

A model for dinitrogen binding in the E4 state of nitrogenase.

ABSTRACT: Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic iron-molybdenum-sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N2-binding E4 state is not known. In this study we present theoretical QM/MM calculations of new structural models of the E4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic redox state. We propose two models as possible candidates for the E4 state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe2 and Fe6 with a terminal sulfhydryl group on either Fe2 or Fe6 (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe2 and Fe6. These structures appear consistent with the bridging hydride proposal put forward from ENDOR studies and are calculated to be lower in energy than other proposed models for E4 at the TPSSh-QM/MM level of theory. We critically analyze the DFT method dependency in calculations of FeMoco that has resulted in strikingly different proposals for this state. Importantly, dinitrogen binds exothermically to either Fe2 or Fe6 in our models, contrary to others, an effect rationalized via the unique ligand field (from the hydrides) at the Fe with an empty coordination site. A low-spin Fe site is proposed as being important to N2 binding. Furthermore, the geometries of these states suggest a feasible reductive elimination step that could follow, as experiments indicate. Via this step, two electrons are released, reducing the cofactor to yield a distorted 4-coordinate Fe2 or Fe6 that partially activates N2. We speculate that stabilization of an N2-bound Fe(i) at Fe6 (not found for Fe2 model) via reductive elimination is a crucial part of N2 activation in nitrogenases, possibly aided by the apical heterometal ion (Mo or V). By using protons from the sulfhydryl group (to regenerate the sulfide bridge between Fe2 and Fe6) and the nearby homocitrate hydroxy group, we calculate a plausible route to yield a diazene intermediate. This is found to be more favorable with the Fe6-bound model than the Fe2-bound model; however, this protonation is uphill in energy, suggesting protonation of N2 might occur later in the catalytic cycle or via another mechanism.

SUBMITTER: Thorhallsson AT

PROVIDER: S-EPMC7069239 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A model for dinitrogen binding in the E4 state of nitrogenase.

Thorhallsson Albert Th AT Benediktsson Bardi B Bjornsson Ragnar R

Chemical science 20191015 48

Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic iron-molybdenum-sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N2-binding E4 state is not known. In this study we present theoretical QM/MM calculations of new structural models of the E4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic ...[more]

PMID: 32206260

Similar Datasets

Project description: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.

Project description:We here report the first direct evidence addressing the possible involvement of Mo in substrate interactions during catalytic turnover. When the alpha-70(Ile) MoFe protein is freeze-trapped during H(+) reduction under Ar, the majority of the resting state EPR signal from the molybdenum-iron cofactor (FeMo-co) disappears and is replaced by the S = 1/2 signal of an intermediate that has been shown to be the E(4) MoFe state, which is activated for N(2) binding and reduction through the accumulation of 4 electrons/protons by FeMo-co. ENDOR studies of E(4) showed that it contains two hydrides bound to FeMo-co. We calculate that Mo involvement in hydride binding would require a vector-coupling coefficient for Mo of |K(Mo)| approximately > 0.2 and determine K(Mo) for the E(4) intermediate state through 35 GHz ENDOR measurements of a (95)Mo enriched MoFe protein, further comparing the results with those for the E(0) resting state. The experiments show that Mo of the resting-state FeMo-co is perturbed by the alpha-70(Ile) substitution and that the isotropic (95)Mo hyperfine coupling in E(4) is a(iso) approximately 4 MHz, less than that for the resting state. The decrease in a(iso) for (95)Mo of E(4) from the already small value in the resting state MoFe protein strongly suggests that the resting Mo(IV) is not one-electron reduced during the accumulation of the four electrons of E(4). In any case, the effective K for Mo is very small; |K(Mo)| approximately < 0.04, at least 5-fold less than the lower bound required for Mo to be involved in forming a Mo-H-Fe, hydride. As the hydride couplings also are both far too small and of the wrong symmetry to be associated with a terminal hydride on Mo, we may thus conclude that Mo does not participate in binding a hydride of the catalytically central E(4) intermediate and that only Fe ions are involved. Nonetheless, the response of the Mo coupling to subtle conformational changes in E(0) and to the formation of E(4) suggests that Mo is intimately involved in tuning the geometric and electronic properties of FeMo-co in these states.

Dataset Information

A model for dinitrogen binding in the E4 state of nitrogenase.

Publications

A model for dinitrogen binding in the E<sub>4</sub> state of nitrogenase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets