Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii

Duyvis, M.G.; Wassink, Hans; Haaker, H.

doi:10.1074/jbc.271.47.29632

Cited by 19 publications

(18 citation statements)

References 26 publications

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“…Following docking, it is expected that conformational changes (probably a series of changes) occur before the electron transfer event (18) which can be characterized by the equilibrium constant K c ) k c /k -c . This adiabatic step is rate-limiting, explaining why the Marcus formalism is not applicable.…”

Section: Discussionmentioning

confidence: 99%

Electron Transfer in Nitrogenase Analyzed by Marcus Theory: Evidence for Gating by MgATP

1998

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Nitrogenase-catalyzed substrate reduction reactions require electron transfer between two component proteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein, in a reaction that is coupled to the hydrolysis of MgATP. In the present work, electron transfer (Marcus) theory has been applied to nitrogenase electron transfer reactions to gain insights into possible roles for MgATP in this reaction. Evidence is presented indicating that an event associated with either MgATP binding or hydrolysis acts to gate electron transfer between the two component proteins. In addition, evidence is presented that the reaction mechanism can be fundamentally changed such that electron transfer becomes rate-limiting by the alteration of a single amino acid within the nitrogenase Fe protein (deletion of Leu 127, L127 Delta). These studies utilized the temperature dependence of intercomponent electron transfer within two different nitrogenase complexes: the wild-type nitrogenase complex that requires MgATP for electron transfer and the L127 Delta Fe protein-MoFe protein complex that does not require MgATP for electron transfer. It was found that the wild-type nitrogenase electron transfer reaction did not conform to Marcus theory, suggesting that an adiabatic event associated with MgATP interaction precedes electron transfer and is rate-limiting. Application of transition state theory provided activation parameters for this rate-limiting step. In contrast, electron transfer from the L127 Delta Fe protein variant to the MoFe protein (which does not require MgATP hydrolysis) was found to be described by Marcus theory, indicating that electron transfer was rate-limiting. Marcus parameters were determined for this reaction with a reorganization energy (lambda) of 2.4 eV, a coupling constant (HAB) of 0.9 cm-1, a free energy change (Delta G' degrees ) of -22.0 kJ/mol, and a donor-acceptor distance (r) of 14 A. These values are consistent with parameters deduced for electron transfer reactions in other protein-protein systems where electron transfer is rate-limiting. Finally, the electron transfer reaction within the L127 Delta Fe protein-MoFe protein complex was found to be reversible. These results are discussed in the context of models for how MgATP interactions might be coupled to electron transfer in nitrogenase.

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Section: Discussionmentioning

confidence: 99%

Electron Transfer in Nitrogenase Analyzed by Marcus Theory: Evidence for Gating by MgATP

1998

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“…The original stopped-flow spectroscopic studies supported a model where MgATP hydrolysis preceded electron transfer. 15 More recent stoppedflow studies indicate that electron transfer precedes MgATP hydrolysis, 55,56 and this possibility was supported by characterization of the altered Fe protein that has switch II shortened. 57 This protein is the same one that is locked into a MgATP-like conformation even in the absence of MgATP and also forms a tight complex when mixed with the MoFe protein.…”

Section: Electron Transfer Can Occur Without Mgatp Hydrolysismentioning

confidence: 96%

Role of Nucleotides in Nitrogenase Catalysis

1997

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“…The mechanism of self-oxidation is still not understood and might not be physiologically relevant. Regardless, it needs to be taken into account in kinetics studies, as it significantly influences nitrogenase reactions performed in vitro with DT …”

Section: Mo N2asementioning

confidence: 99%

“…Regardless, it needs to be taken into account in kinetics studies, as it significantly influences nitrogenase reactions performed in vitro with DT. 375 4.1.2. FeP red .…”

Section: Fep and The F-clustermentioning

confidence: 99%

The Spectroscopy of Nitrogenases

et al. 2020

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Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron–sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

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Pre-steady-state Kinetics of Nitrogenase from Azotobacter vinelandii

Cited by 19 publications

References 26 publications

Electron Transfer in Nitrogenase Analyzed by Marcus Theory: Evidence for Gating by MgATP

Electron Transfer in Nitrogenase Analyzed by Marcus Theory: Evidence for Gating by MgATP

Role of Nucleotides in Nitrogenase Catalysis

The Spectroscopy of Nitrogenases

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