J. Phys. Chem. B 2008, 112, 11435–11439
11435
Exploring the Interstitial Atom in the FeMo Cofactor of Nitrogenase: Insights from QM and QM/MM Calculations Hujun Xie, Ruibo Wu, Zhaohui Zhou, and Zexing Cao* Department of Chemistry and State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen UniVersity, Xiamen 361005, China ReceiVed: April 24, 2008; ReVised Manuscript ReceiVed: June 22, 2008
Density functional theory and combined quantum mechanics and molecular mechanics (QM/MM) calculations have been used to explore structural features of the FeMo cofactor with an interstitial atom X (X ) N, C, or O) and its interactions with CO and N2. Predicted frequencies of the metal-bound CO, QM/MM-optimized geometries, and calculated redox potentials of the FeMo cofactor with different central ligands show that the oxygen atom is the candidate for the interstitial atom. Calculations on the interactions of the FeMo cofactor with CO and N2 reveal that there is a remarkable dependence of the binding energy on the binding site and the interstitial atom. Generally, the Fe2 site of the FeMo cofactor has stronger interactions with CO and N2 than Fe6, and both the Fe2 and Fe6 sites in the N-centered and O-centered clusters of the FeMo cofactor can effectively bind N2 while the coordination of N2 to the Fe6 site of the C-centered active cluster is unfavorable energetically. Present results indicate that the protein environment is important for computational characterization of the structure of the FeMo cofactor and properties of the metal-bound CO and N2 are sensitive to the interstitial atom. 1. Introduction Nitrogenases, widely present in diverse diazotrophic microorganisms,1-3 are responsible for the catalytic reduction of N2 to NH3 under ambient conditions. The Mo-dependent nitrogenase comprising the iron (Fe) protein and the molybdenum-iron (MoFe) protein was found to have the highest activity among known nitrogenases. The MoFe protein contains the FeMo cofactor (FeMoco) as the catalytic active site for binding and reduction of substrate. During catalysis, the binding of MgATP to the Fe protein and subsequent hydrolysis play an important role in driving the electron transfer from the Fe protein to the MoFe protein.4-6 Recent X-ray crystallographic study of the FeMo protein with a high resolution of 1.16 Å revealed an unidentified light atom (X) inside the central Fe6 triangular prism of FeMoco,7 denoted as FeMoco(µ6-X). The single N, O, or C atom was assumed as a possible candidate for the interstitial atom X. Following density functional calculations with different cluster models generally ruled out the possibility of oxygen atom as an interstitial atom due to a remarkable expansion of the O-centered active cluster, while the nitrogen atom was most likely to be a central atom.8-11 In recent theoretical studies on the mechanism of biological nitrogen fixation,12-15 the N-centered cluster model of the FeMo cofactor has been widely used. However, ENDOR and ESEEM studies argued against the possibility of nitrogen as the interstitial atom.16 More recently, ENDOR and ESEEM measurements in combination with the broken-symmetry (BS) density functional calculations of hyperfine coupling parameters for Fe and for X revealed that X * N/C,17 unless X in effect is magnetically decoupled from the resting FeMoco electron spin system. Clearly, the identification of the interstitial atom remains open and further theoretical * Corresponding author. Fax: + 86 -592-2183047. E-mail: zxcao@ xmu.edu.cn.
calculations and higher resolution X-ray structural measurements are highly required to verify the unsolved central atom X. Here we conducted extensive QM and QM/MM calculations on the FeMo cofactor of nitrogenase and its interactions with CO and N2. The applicability of different functionals in description of the FeMo cofactor has been evaluated. The effect of the protein environment on the geometry of the FeMo cofactor has been discussed on the basis of QM and combined QM/MM calculations. 2. Computational Methods QM and QM/MM models for the FeMoco of nitrogenase are shown in Figure 1. The QM mode of FeMoco includes the [Mo7Fe-9S(µ6-X)] cluster, the side ligands of a methyl thiolate for Cys275, an imidazole for His442, and homocitrate, as well as water molecules involved in the hydrogen-bond network connecting to the active cluster, i.e. [Mo7Fe9S(µ6-X)(SCH3)(imidazole)(homocitrate)]-q · 12H2O (q ) -4, -5, and -6 for O, N, and C, respectively). The PW91,18 RPBE,19 and BLYP20,21 functionals have been considered in the spin-unrestricted Kohn-Sham computations with the all-electron double numerical basis set plus polarization functions implemented in DMol3 program.22 The lowest-energy spin coupling state23 of the resting state with S ) 3/2 for the FeMoco(µ6-X) cluster was considered in the calculation. In the QM/MM calculations, the complete computational model was constructed by solvating the crystal structure (PDB code: 1M1N)7 into a sphere of water molecules with a radius of 30 Å and then carrying out molecular dynamic (MD) simulations at the molecular mechanics (MM) level using CHARMM program24 for 200 ps to bring the system to an equilibrated state. During MM-based MD simulations, the FeMoco(µ6-X) cluster was frozen for simplification. This model contains 13768 atoms including the FeMoco(µ6-X) cluster, 477 amino acids, and 2068 water molecules. The QM/MM calcula-
10.1021/jp803616z CCC: $40.75 2008 American Chemical Society Published on Web 08/16/2008
11436 J. Phys. Chem. B, Vol. 112, No. 36, 2008
Xie et al.
Figure 1. QM and QM/MM models used in calculations.
TABLE 1: Average Bond Lengths (Å) and Angles (deg) of the FeMoco(µ6-X) Cluster by QM/MM Calculations PW-LDA a
TABLE 2: Average Bond Lengths (in Å) and Angles (in deg) of the FeMoco(µ6-X) Cluster by QM Calculations
PBE
N3-
PW91
separation/angle
exptb
N3-
C4-
O2-
N3-
C4-
O2-
separation/anglea
exptb
N3-
O2-
C4-
vacancy RPBE BLYP
Fe′-X Fe′′-X ∠Fe′-X-Fe′′ Fe-Fe′ Fe′-Fe′ Fe′-Fe′′ Fe′′-Fe′′ Fe′′-Mo Fe′-Fe′′ diagonal Fe-X
1.98 2.02 80.9 2.67 2.65 2.59 2.61 2.69 3.70 meanc max mean max
1.97 1.91 82.9 2.63 2.50 2.57 2.53 2.68 3.60 0.06 -0.10 0.07 -0.17
1.95 1.92 83.1 2.64 2.51 2.57 2.51 2.67 3.59 0.07 -0.09 0.07 -0.15
2.05 1.99 85.0 2.63 2.56 2.73 2.60 2.69 3.76 0.05 0.11 0.06 0.18
2.01 2.03 82.2 2.77 2.67 2.68 2.65 2.78 3.77 0.02 0.15 0.06 0.16
2.09 1.93 82.3 2.76 2.64 2.66 2.60 2.76 3.73 0.10 0.16 0.06 0.16
2.08 2.17 83.3 2.78 2.93 2.82 2.53 2.74 3.91 0.13 0.22 0.16 0.32
Fe′-X Fe′′-X
