Selenol binds to iron in nitrogenase iron-molybdenumcofactor: an extended x-ray absorption fine structure study.

Abstract

The biological N2-flxation reaction is catalyzed by the enzyme nitrogenase. The metal cluster active site of this enzyme, the iron-molybdenum cofactor (FeMoco), can be studied either while bound within the MoFe protein component of nitrogenase or after it has been extracted into N-methylformamide. The two species are similar but not identical. For example, the addition ofthiophenol or selenophenol to isolated FeMoco causes its rather broad S 3/2 electron paramagetic resonance signal to sharpen and more closely approach the signal exhibited by protein-bound FeMoco. The nature of this thiol/selenol binding site has been investigated by using Se-K edge extended x-ray absorption frne structure (EXAFS) to study selenophenol ligated to FeMoco, and the results are reported here. EXAFS data analysis at the ligand Se-K edge was performed with a set of software, GNXAS, that provides for direct calculation ofthe theoretical EXAFS signals and least-squares fits to the experimental data. Data analysis results show definitively that the selenol (and by inference thiol) binds to Fe at a distance of 2.4 A. In contrast, unacceptable fits are obtained with either Mo or S as the liganded atom (instead of Fe). These results provide quantitative details about an exchangeable thiol/selenol binding site on FeMoco in its isolated, solution state and establish an Fe atom as the site of this reaction. Furthermore, the utility of ligand-based EXAFS as a probe of coordination in polynuclear metal clusters is demonstrated.The biological N2-fixation reaction is catalyzed by the enzyme nitrogenase. The active site of this enzyme is a metal cluster of stoichiometry Mo:Fe7:.

X‐ray Absorption Spectroscopy Study of Zinc Coordination in Tetanus Neurotoxin, Astacin, Alkaline Protease and Thermolysin

“…First of all we note that all four Fourier transforms have an enhanced peak around 0.35 nm. It is well known that an enhancement in the amplitude of this peak, when found in conjunction with nitragcns in the first shell, is normally attributed to niultiple scattering effects from the imidazole rings of the cciordinated histidines (Bunker et al, 1982;Guriiian et al, 1986;Westre et al, 1994;Conradson et al, 1994;Blackburn et al, 1983). (Single scattering contributions froin a inore distant second shell also contribute to the magnitude of the peak.)…”

“…In principle the near-edge region of the XAS spectrum contains detailed information on the coordination gconielry around the absorber (Westre et al, 1994;Conradson et al, 1994). Below threshold there can be transitions to bound states with line-like features.…”

Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior

“…This well-known experiment was followed using EPR spectroscopy, and established the 1:1 stoichiometry of the reaction. The reaction has been further investigated using 19 F NMR with p-tri¯uoromethyl-benzenethiol as the reporter molecule [4,51], and by titration with benzeneselenol using both K-edge EXAFS spectroscopy [15,49,50] and EPR [15]. Nevertheless, an analysis of the EPR titration data as a simple equilibrium binding reaction, in analogy to reaction 2 above, has not been successful.…”

“…The S=3/2 EPR spectrum of FeMoco(sr) in NMF solution titrates with stoichiometric thiol [8,15,48], which is known to bind at an iron site [15,49,50] on this cluster. Thiol binding has more recently been used as a kinetic probe into FeMoco(sr) reactivity [13,44].…”

Expression and Association of Group IV Nitrogenase NifD and NifH Homologs in the Non-Nitrogen-Fixing ArchaeonMethanocaldococcus jannaschii

“…Residing at the ␣/␤ subunit interface of dinitrogenase is an unusual [Fe 8 S 7 ] cluster called the P cluster (7,42,51), which has been shown to transfer electrons and protons to the active site [Fe 7 S 8 MoN(homocitrate)] cluster (called FeMo-co), which is the active site of substrate reduction. FeMo-co lies within the ␣ subunit (NifD) (8,12,26,27,29,36,54).…”