| Autor: |
Lukoyanov DA; Department of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA., Yang ZY; Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA., Shisler K; Institute of Biological Sciences, Washington State University, Pullman, Washington, 99164, USA., Peters JW; Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, 73019, USA., Raugei S; Physical and Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, 99352, USA., Dean DR; Biochemistry Department, Virginia Tech, Blacksburg, Virginia, 24061, USA., Seefeldt LC; Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA., Hoffman BM; Department of Chemistry and Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA. |
| Abstrakt: |
Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe 7 S 9 MoC-homocitrate) as a critical N 2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E 4 (4H), which has accumulated 4[e - /H + ] as two bridging hydrides, Fe2-H-Fe6 and Fe3-H-Fe7, and protons bound to two sulfurs. E 4 (4H) is poised to bind/reduce N 2 as driven by mechanistically-coupled H 2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H 2 as the enzyme relaxes to state E 2 (2H), containing 2[e - /H + ] as a hydride and sulfur-bound proton; accumulation of E 4 (4H) in α-V70I is enhanced by HP suppression. EPR and 95 Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e - /H + ] to the E 0 state of the WT MoFe protein and to both α-V70I conformations generating E 2 (2H) that contains the Fe3-H-Fe7 bridging hydride; accumulation of another 2[e - /H + ] generates E 4 (4H) with Fe2-H-Fe6 as the second hydride. E 4 (4H) in WT enzyme and a minority α-V70I E 4 (4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2-H-Fe6 followed by slower HP of Fe3-H-Fe7, which leads to transient accumulation of E 2 (2H) containing Fe3-H-Fe7. In the dominant α-V70I E 4 (4H) conformation, HP of Fe2-H-Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3-H-Fe7 occurs first and the resulting E 2 (2H) contains Fe2-H-Fe6. It is this HP suppression in E 4 (4H) that enables α-V70I MoFe to accumulate E 4 (4H) in high occupancy. In addition, HP suppression in α-V70I E 4 (4H) kinetically unmasks hydride reductive-elimination without N 2 -binding, a process that is precluded in WT enzyme. |
