Tyrosine-Coordinated P-Cluster in G. diazotrophicus Nitrogenase: Evidence for the Importance of O-Based Ligands in Conformationally Gated Electron Transfer


JACS August 2016: Abstract

Cedric P. Owens, Faith E. H. Katz, Cole H. Carter, Victoria F. Oswald, and F. Akif Tezcan

    The P-cluster is a unique iron–sulfur center that likely functions as a dynamic electron (e–) relay site between the Fe-protein and the catalytic FeMo-cofactor in nitrogenase. The P-cluster has been shown to undergo large conformational changes upon 2-e– oxidation which entail the coordination of two of the Fe centers to a Ser side chain and a backbone amide N, respectively. Yet, how and if this 2-e– oxidized state (POX) is involved in catalysis by nitrogenase is not well established. Here, we present the crystal structures of reduced and oxidized MoFe-protein (MoFeP) from Gluconacetobacter diazotrophicus (Gd), which natively possesses an Ala residue in the position of the Ser ligand to the P-cluster. While reduced Gd-MoFeP is structurally identical to previously characterized counterparts around the FeMo-cofactor, oxidized Gd-MoFeP features an unusual Tyr coordination to its P-cluster along with ligation by a backbone amide nitrogen. EPR analysis of the oxidized Gd-MoFeP P-cluster confirmed that it is a 2-e– oxidized, integer-spin species. Importantly, we have found that the sequence positions corresponding to the Ser and Tyr ligands are almost completely covariant among Group I nitrogenases. These findings strongly support the possibility that the POX state is functionally relevant in nitrogenase catalysis and that a hard, O-based anionic ligand serves to stabilize this state in a switchable fashion.

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Nature (May 2016): Abstract

Yuta Suzuki, Giovanni Cardone, David Restrepo, Pablo D. Zavattieri, Timothy S. Baker & F. Akif Tezcan

    Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties that make them highly attractive in many applications. Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers, bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development. More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour. As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C4-symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants (C98RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. C98RhuA lattices display a Poisson’s ratio of −1 — the lowest thermodynamically possible value for an isotropic material—making them auxetic.

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Evidence for Functionally Relevant Encounter Complexes in Nitrogenase Catalysis


JACS September 2015: Abstract

Cedric P. Owens, Faith E. H. Katz, Cole H. Carter, Maria A. Luca, and F. Akif Tezcan

    Nitrogenase is the only enzyme that can convert atmospheric dinitrogen (N2) into biologically usable ammonia (NH3). To achieve this multielectron redox process, the nitrogenase component proteins, MoFe-protein (MoFeP) and Fe-protein (FeP), repeatedly associate and dissociate in an ATP-dependent manner, where one electron is transferred from FeP to MoFeP per association. Here, we provide experimental evidence that encounter complexes between FeP and MoFeP play a functional role in nitrogenase catalysis. The encounter complexes are stabilized by electrostatic interactions involving a positively charged patch on the β-subunit of MoFeP. Three single mutations (βAsn399Glu, βLys400Glu, and βArg401Glu) in this patch were generated in Azotobacter vinelandii MoFeP. All of the resulting variants displayed decreases in specific catalytic activity, with the βK400E mutation showing the largest effect. As simulated by the Thorneley–Lowe kinetic scheme, this single mutation lowered the rate constant for FeP-MoFeP association 5-fold. We also found that the βK400E mutation did not affect the coupling of ATP hydrolysis with electron transfer (ET) between FeP and MoFeP. These data suggest a mechanism where FeP initially forms encounter complexes on the MoFeP β-subunit surface en route to the ATP-activated, ET-competent complex over the αβ-interface.

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