Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase

Spatzal, Thomas; Perez, Kathryn; Einsle, Oliver; Howard, James B.; Rees, Douglas C.

doi:10.1126/science.1256679

Cited by 375 publications

(491 citation statements)

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“…Strikingly, the signal intensities of these samples displayed a linear correlation with the amounts of CO released from them ( Fig. 3C), firmly establishing CO as the origin of the unique EPR To carry out experiments on the catalytic competence of the CO-bound conformations, the VFe protein was loaded with 12 CO or 13 CO in the presence of Eu II -DTPA, reisolated from the CO atmosphere, and subjected to turnover conditions upon addition of Fe protein and ATP in the presence of dithionite. EPR analysis revealed that the VFe protein preloaded with 12 CO (Fig.…”

Section: Resultsmentioning

confidence: 66%

“…2A) (12). Electron paramagnetic resonance (EPR) analysis of this CO-bound MoFe protein identified a CO-derived signal (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…Fortunately, such a quest has already had an excellent start, owing a great deal to the recent crystal structure of CO-bound MoFe protein (12). Despite the fact that the CO-bound MoFe protein is hardly capable of turnover (and hence the designation CO-inhibited MoFe protein), its close resemblance to the CObound VFe protein renders it highly relevant to the catalytic turnover of CO.…”

Section: Discussionmentioning

confidence: 99%

“…Samples were prepared according to the published method that was used to generate a CObound form of MoFe protein for crystallographic analysis (12). In short, the MoFe protein was equilibrated with 100% (or 1 atm) CO before replacement of 10% headspace by an equal volume of C 2 H 2 .…”

Section: Methodsmentioning

confidence: 99%

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Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

Lee

Fay

Weng

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

100

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Biocatalysis by nitrogenase, particularly the reduction of N 2 and CO by this enzyme, has tremendous significance in environmentand energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N 2 reduction.nitrogenase | vanadium | carbon monoxide | turnover | substrate binding N itrogenases are complex metalloenzymes that catalyze the reduction of a variety of substrates under ambient conditions (1-3). Among them, two reactions bear significant relevance to environment-and energy-related areas: (i) the reduction of dinitrogen (N 2 ), a key element of nitrogen cycle in our biosphere, to the bio-accessible form of ammonia (NH 3 ); and (ii) the reduction of carbon monoxide (CO), a waste product from car and factory exhausts, to useful hydrocarbon products. The molybdenum (Mo)-and vanadium (V)-nitrogenases are two homologous members of this enzyme family. Both enzymes consist of a reductase component (nifH-or vnfH-encoded Fe protein) and a catalytic component (nifDK-encoded MoFe protein or vnfDGK-encoded VFe protein). Substrate turnover by both nitrogenases involves the formation of a functional complex between the two component proteins (1-3), which enables adenosine triphosphate (ATP)-dependent, interprotein transfer of electrons from the reductase component to the cofactor site of the catalytic component for the subsequent reduction of substrates.Designated the M and V cluster, respectively, the cofactors of Mo-and V-nitrogenases closely resemble each other in geometry (3-7). Previous Fe K-edge X-ray absorption spectroscopy (XAS)/ extended X-ray absorption fine structure (EXAFS) analysis revealed that the two cofactors had nearly indistinguishable metalsulfur core structures, each comprising MFe 3 S 3 (M = Mo or V) and Fe 4 S 3 subclusters bridged by three μ 2 -coordinated, belt-sulfur (S) atoms ( Fig. 1 A and B) (3, 6, 8). Recently, we performed a K-valence X-ray emission spectroscopy (XES) study of both protein-bound and solvent-extracted V clusters (Fig. 1C), which identified a carbide (C 4-)-specific XES feature of the V cluster that was observed earlier in the case of the M cluster (9). Thus, the two cofactors not only share an overall homology in structure, but also have the same inner strengths that originate from the μ 6 -coordinated interstitial carbide. Surprisingly, despite the significant homology bet...

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Section: Resultsmentioning

confidence: 66%

“…2A) (12). Electron paramagnetic resonance (EPR) analysis of this CO-bound MoFe protein identified a CO-derived signal (Fig.…”

Section: Resultsmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

Lee

Fay

Weng

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

100

View full text Add to dashboard Cite

show abstract

“…Preservation of these features may be crucial for the reactivity of Fe 6 RHH , as a recent crystallographic study revealed displacement of a "belt" S atom by a CO moiety upon binding of CO to the M-cluster, [22] an event requiring the presence of the interstitial C atom to maintain the structural integrity of the M-cluster when the S "belt" undergoes significant rearrangement during catalysis. Thus, by analogy, the equivalents to the "belt" S and "central" C atoms in Fe 6 RHH may render it capable of interacting with substrates in an analogous manner to that of the M-cluster; in particular, such an analogy could explain the reactivity of Fe 6 RHH toward CN − , which parallels the reactivity of the Mcluster toward CO (an isoelectronic molecule to the CN − ion), in reductive C-C coupling.…”

Section: Introductionmentioning

confidence: 99%

Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme

et al. 2015

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Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe 7 S 9 C] n− ; designated M-cluster). Here, we report the formation of an artificial, nitrogenase-mimicking enzyme upon insertion of a synthetic model complex ([Fe 6 S 9 (SEt) 2 ] 4− ; designated Fe 6 RHH ) into the catalytic component of nitrogenase (designated NifDK). Two Fe 6 RHH clusters were inserted into NifDK, rendering the resultant protein (designated NifDK Fe ) in a similar conformation to that upon insertion of native M-clusters. NifDK Fe could work together with the reductase component of nitrogenase to reduce C 2 H 2 in an ATP-dependent reaction. It could also act as an enzyme on its own in the presence of Eu(II) DTPA, displaying a strong activity in C 2 H 2 reduction while demonstrating an ability to reduce CN − to C1-C3 hydrocarbons in an ATP-independent manner.

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Nitrogen Fixation

Newton

2015

Kirk-Othmer Encyclopedia of Chemical Technology

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About 80% of the Earth's atmosphere is made up of nitrogen gas (N 2 ), but nitrogen is most often the limiting nutrient for agricultural productivity, because neither plants nor animals can use it directly. It must first be fixed into either ammonium or nitrate. Only the simplest life forms, bacteria and archaea, can produce ammonium from N 2 using the enzyme called nitrogenase. N 2 is also fixed by nonbiological processes, which contribute equally to the total annual fixation rate. In fact, without commercial fertilizers, the human population could not be supported. This article first describes the major industrial processes and their shortcomings, emphasizing the dominant Haber‐Bosch process. Then, an overview of the various biological nitrogen‐fixing systems, both free‐living and symbiotic organisms, focusing on the agriculturally important legume– Rhizobium symbiosis is provided. Nitrogenase is detailed and analyzed next with sections on its evolution, biosynthesis, composition and structure, regulation, and catalytic mechanism, the last of which is now beginning to be understood in some detail. The penultimate section describes attempts to duplicate either the reactivity or the structure of nitrogenase in chemical systems, some of which have achieved limited catalytic capability. Finally, the last section attempts to highlight both the important strides made and the problems that remain. These include limiting losses of applied fertilizer to ground water and atmosphere; careful matching of inoculant with cultivar; reducing fossil fuel energy inputs to fertilizer production; manipulating plant colonization and symbiosis development to produce new pairings; and simplifying the biological process for transfer to other organisms.

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Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase

Cited by 375 publications

References 44 publications

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase

Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme

Nitrogen Fixation

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