X-ray Absorption and Resonance Raman Studies of Methyl-Coenzyme M Reductase Indicating That Ligand Exchange and Macrocycle Reduction Accompany Reductive Activation

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• 作者：Qun Tang ; Paul E. Carrington ; Yih-Chern Horng ; Michael J. Maroney ; Stephen W. Ragsdale ; and David F. Bocian
• 刊名：Journal of the American Chemical Society
• 出版年：2002
• 出版时间：November 6, 2002
• 年：2002
• 卷：124
• 期：44
• 页码：13242 - 13256
• 全文大小：337K
• 年卷期：v.124,no.44(November 6, 2002)
• ISSN：1520-5126

文摘

Methyl-coenzyme M reductase (MCR) catalyzes methane formation from methyl-coenzyme M(methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). MCR contains a nickel hydrocorphin cofactor at its active site, called cofactor F₄₃₀. Here we present evidence that the macrocyclic ligandparticipates in the redox chemistry involved in catalysis. The active form of MCR, the red1 state, is generatedby reducing another spectroscopically distinct form called ox1 with titanium(III) citrate. Previous electronparamagnetic resonance (EPR) and ¹⁴N electron nuclear double resonance (ENDOR) studies indicate thatboth the ox1 and red1 states are best described as formally Ni(I) species on the basis of the character ofthe orbital containing the spin in the two EPR-active species. Herein, X-ray absorption spectroscopic (XAS)and resonance Raman (RR) studies are reported for the inactive (EPR-silent) forms and the red1 and ox1states of MCR. RR spectra are also reported for isolated cofactor F₄₃₀ in the reduced, resting, and oxidizedstates; selected RR data are reported for the ¹⁵N and ⁶⁴Ni isotopomers of the cofactor, both in the intactenzyme and in solution. Small Ni K-edge energy shifts indicate that minimal electron density changesoccur at the Ni center during redox cycling of the enzyme. Titrations with Ti(III) indicate a 3-electron reductionof free cofactor F₄₃₀ to generate a stable Ni(I) state and a 2-electron reduction of Ni(I)-ox1 to Ni(I)-red1.Analyses of the XANES and EXAFS data reveal that both the ox1 and red1 forms are best described ashexacoordinate and that the main difference between ox1 and red1 is the absence of an axial thiolateligand in the red1 state. The RR data indicate that cofactor F₄₃₀ undergoes a significant conformationalchange when it binds to MCR. Furthermore, the vibrational characteristics of the ox1 state and red1 statesare significantly different, especially in hydrocorphin ring modes with appreciable C=N stretching character.It is proposed that these differences arise from a 2-electron reduction of the hydrocorphin ring uponconversion to the red1 form. Presumably, the ring-reduction and ligand-exchange reactions reported hereinunderlie the enhanced activity of MCR_red1, the only form of MCR that can react productively with the methylgroup of methyl-SCoM.