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.
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Incorporation of an Asymmetric Mo−Fe−S Cluster as an Artificial Cofactor into Nitrogenase
Nitrogenase employs a sophisticated electron transfer system and a Mo−Fe−S−C cofactor, designated the M‐cluster [(cit)MoFe7S9C]), to reduce atmospheric N2to bioaccessible NH3. Previously, we have shown that the cofactor‐free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe−S cluster [Fe6S9(SEt)2]4−. Here, we demonstrate the utility of an asymmetric Mo−Fe−S cluster [Cp*MoFe5S9(SH)]3−as an alternative artificial cofactor upon incorporation into the cofactor‐free nitrogenase scaffold. The resultant semi‐artificial enzyme catalytically reduces C2H2to C2H4, and CN−into short‐chain hydrocarbons, yet it is clearly distinct in activity from its [Fe6S9(SEt)2]4−‐reconstituted counterpart, pointing to the possibility to employ molecular design and cluster synthesis strategies to further develop semi‐artificial or artificial systems with desired catalytic activities.
more » « less- NSF-PAR ID:
- 10372835
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 23
- Issue:
- 19
- ISSN:
- 1439-4227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The interest in methyl group C–H bond activation near or bound to iron-containing clusters is of key biological importance, due to the broad relevance of radical SAM reactions. Specifically, such processes are implicated in the biogenesis of the interstitial carbide found in the nitrogenase FeMoco active site. In this work, we find that the diamagnetic, methyl-thiolate capped iron–carbonyl cluster anion [(CH 3 S)Fe 3 (CO) 9 ] − (1) undergoes facile C–H hydrogen atom abstraction upon treatment with TEMPO. The process leads to (i) eradication of the CH 3 moiety, (ii) formation of a sulfide bridge, and (iii) cluster dimerization—thereby generating the ‘dimer of trimers’ cluster [K(benzo-15-crown-5) 2 ] 2 [(SFe 2 (CO) 12 ) 2 Fe(CO) 2 ] (2). In contrast, the corresponding isopropyl variant [Fe 3 (S i Pr)(CO) 9 ] − (3) does not react with TEMPO . Mass spectrometry confirmed the presence of TEMPOH, as well as CO oxidation vis a vis CO 2 and 2,2,6,6-tetramethylpiperidine. GC-MS measurements of the headspace reveal that the ultimate fate of the methyl carbon is likely incorporation into multiple products—one of which may be a volatile low mass hydrocarbon—rather than carbon/carbide incorporation.more » « less
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Abstract The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox‐active [Fe4S4] cluster. Here we report the synthesis and characterization of a water‐soluble [Fe4Se4] cluster that is used to substitute the [Fe4S4] cluster of the
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Abstract The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox‐active [Fe4S4] cluster. Here we report the synthesis and characterization of a water‐soluble [Fe4Se4] cluster that is used to substitute the [Fe4S4] cluster of the
Azotobacter vinelandii Fe protein (Av NifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4Se4] cluster to adopt the super‐reduced, all‐ferrous state upon its incorporation intoAv NifH. Moreover, these studies reveal that the [Fe4Se4] cluster inAv NifH already assumes a partial all‐ferrous state ([Fe4Se4]0) in the presence of dithionite, where its [Fe4S4] counterpart inAv NifH exists solely in the reduced state ([Fe4S4]1+). Such a discrepancy in the redox properties of theAv NifH‐associated [Fe4Se4] and [Fe4S4] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen‐substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.
