Docking and Migration of Carbon Monoxide in Nitrogenase: The Case for Gated Pockets from Infrared Spectroscopy and Molecular Dynamics

Gee, Leland B.; Leontyev, Igor; Stuchebrukhov, Alexei A.; Scott, Aubrey D.; Pelmenschikov, Vladimir; Cramer, Stephen P.

doi:10.1021/acs.biochem.5b00216

Cited by 24 publications

(17 citation statements)

References 58 publications

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“…The different results for CowN protection under 0.1 and 0.001 atm CO, in terms of both maximum activity and difference in K D app , can be interpreted based on how CO diffuses through nitrogenase to reach FeMoco. As mentioned previously, CO has more than one access pathway to FeMoco and several binding modes ( 5 , 48 , 49 ). The CowN dose response is consistent with a model in which CowN protects nitrogenase efficiently from low concentrations of CO by blocking the preferred CO access channel and/or altering the higher-affinity but not lower-affinity CO-binding sites.…”

Section: Resultsmentioning

confidence: 92%

“…αG69 is located at the terminus of the so-called Igarashi channel (also referred to as IS channel) that starts on the protein surface at residues αK176 and αE263 and later passes by αV70 ( A. vinelandii numbering) ( 5 , 48 ). Subsequently, IR spectroscopy experiments and MD simulations provided further evidence that CO likely migrates through this channel ( 49 ).…”

Section: Resultsmentioning

confidence: 99%

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CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

Medina

Bretzing

Aviles

et al. 2021

Journal of Biological Chemistry

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Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N 2 ) to ammonia (NH 3 ). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN’s protection mechanism involves decreasing the binding affinity of CO to nitrogenase’s active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

Medina

Bretzing

Aviles

et al. 2021

Journal of Biological Chemistry

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“…Figure 1b), derived from a molecular dynamics (MD) calculation using our FeMo-cofactor EFF calculation together with a typical protein force field. The forcefield development and its employment are detailed in our previous work [66]. We find that such a calculation predicts occasional closest approaches of 2.05 Å or less.…”

Section: Resultsmentioning

confidence: 96%

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy — Observation of a candidate promoting vibration

Maiuri

Delfino

Cerullo

et al. 2015

Journal of Inorganic Biochemistry

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We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.2 ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at ~84, 116, 189, and 226 cm−1. Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N2ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226 cm−1 is assigned as a potential ‘promoting vibration in’ the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group.

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“…Cramer et al. explored the photo‐dissociation of CO from FeMo‐co into a nearby docking site about 5 Å from Fe2 and then using molecular dynamics followed the movement of CO into more distant pockets toward the protein surface …”

Section: Components Of the Nitrogenase Mechanismmentioning

confidence: 99%

Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase

Dance

2020

ChemBioChem

103

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Docking and Migration of Carbon Monoxide in Nitrogenase: The Case for Gated Pockets from Infrared Spectroscopy and Molecular Dynamics

Cited by 24 publications

References 58 publications

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy — Observation of a candidate promoting vibration

Computational Investigations of the Chemical Mechanism of the Enzyme Nitrogenase

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