Postbiosynthetic modification of a precursor to the nitrogenase iron–molybdenum cofactor

Srisantitham, Suppachai; Badding, Edward D.; Suess, Daniel L. M.

doi:10.1073/pnas.2015361118

Cited by 12 publications

(15 citation statements)

References 66 publications

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“…[ 77 , 78 ] We note that the models we suggest have distinct molecular and electronic structures that should be spectroscopically distinguishable, if experimental problems associated with low accumulation of reduced FeMoco states can be overcome or if belt Fe‐selective 57 Fe isotope labelling of FeMoco becomes possible. [79] Apart from ENDOR, direct detection of a hydride (or possibly the terminal SH group) through, for example, 57 Fe nuclear resonance vibrational spectroscopy (as has recently been successfully applied to hydrogenase enzymes[ 80 , 81 , 82 , 83 , 84 ]) could become possible and a spectroscopic experiment sensitive to Fe oxidation state (e. g., Mössbauer or Fe X‐ray absorption) should in principle allow a clear distinction between E 2 ‐hyd and E 2 ‐nonhyd models.…”

Section: Discussionmentioning

confidence: 99%

The E₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H₂ Evolution

Thorhallsson

Björnsson

2021

Chemistry A European J

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The iron-molybdenum cofactor (FeMoco) is responsible for dinitrogen reduction in Mo nitrogenase. Unlike the resting state, E 0 , reduced states of FeMoco are much less well characterized. The E 2 state has been proposed to contain a hydride but direct spectroscopic evidence is still lacking. The E 2 state can, however, relax back the E 0 state via a H 2 sidereaction, implying a hydride intermediate prior to H 2 formation. This E 2 !E 0 pathway is one of the primary mechanisms for H 2 formation under low-electron flux conditions. In this study we present an exploration of the energy surface of the E 2 state. Utilizing both cluster-continuum and QM/MM calculations, we explore various classes of E 2 models: including terminal hydrides, bridging hydrides with a closed or open sulfide-bridge, as well as models without. Impor-tantly, we find the hemilability of a protonated belt-sulfide to strongly influence the stability of hydrides. Surprisingly, nonhydride models are found to be almost equally favorable as hydride models. While the cluster-continuum calculations suggest multiple possibilities, QM/MM suggests only two models as contenders for the E 2 state. These models feature either i) a bridging hydride between Fe 2 and Fe 6 and an open sulfide-bridge with terminal SH on Fe 6 (E 2 -hyd) or ii) a double belt-sulfide protonated, reduced cofactor without a hydride (E 2 -nonhyd). We suggest both models as contenders for the E 2 redox state and further calculate a mechanism for H 2 evolution. The changes in electronic structure of FeMoco during the proposed redox-state cycle, E 0 !E 1 !E 2 !E 0 , are discussed.

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Section: Discussionmentioning

confidence: 99%

The E₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H₂ Evolution

Thorhallsson

Björnsson

2021

Chemistry A European J

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“…Having determined the role of βS188 in maintaining the structural and functional integrity of the P-cluster under in vivo conditions, we investigated if the absence of this Ser ligand could also lead to dynamic metal exchange with the environment. We were interested in this possibility not only because it could furnish site-specifically metal-substituted variants of the P-cluster with unusual/useful electronic properties (as shown in the case of FeMoco), , but also because it could help demonstrate that a possible physiological role of βS188 may be to reduce the vulnerability of the P-cluster to mismetallation. We previously determined via X-ray crystallography that the two missing Fe centers in the oxidized βS188A P-cluster could be replenished upon reduction with DT to obtain the fully reconstituted P-cluster .…”

Section: Resultsmentioning

confidence: 99%

Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster

et al. 2022

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Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (P N ) state, the P-cluster is coordinated by six cysteine residues. Upon twoelectron oxidation to the P 2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, βSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redoxlabile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av βSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that βSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of βSer188 both in vivo and in vitro by characterizing the ability of Av βSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of βSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that βSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters.

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“…FeV-cofactor was isolated from pure FeFe protein following procedures previously described for FeMo-cofactor (Srisantitham et al, 2021). Metal content of the isolated cofactor (Fe and V) was determined by inductively coupled plasma mass spectrometry (ICP-MS) (Metal Analysis Service, Virginia Tech).…”

Section: Protein Purification and Analysismentioning

confidence: 99%

AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV‐cofactor

Pérez-González

Jiménez-Vicente

Salinero-Lanzarote

et al. 2022

Molecular Microbiology

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Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N 2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor.Fe-only nitrogenase is a poor catalyst for N 2 fixation, when compared to the Modependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N 2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.

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Postbiosynthetic modification of a precursor to the nitrogenase iron–molybdenum cofactor

Cited by 12 publications

References 66 publications

The E₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H₂ Evolution

The E₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H₂ Evolution

Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster

AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV‐cofactor

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Postbiosynthetic modification of a precursor to the nitrogenase iron–molybdenum cofactor

Cited by 12 publications

References 66 publications

The E2 state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H2 Evolution

The E2 state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H2 Evolution

Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster

AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV‐cofactor

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The E₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H₂ Evolution

The E₂ state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H₂ Evolution