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Ribbe, Markus W.

Publications and source records attributed to Ribbe, Markus W..

12 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Cofactor maturase NifEN: A prototype ancient nitrogenase?

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N 2 at its surface-exposed L-cluster ([Fe 8 S 9 C]), a structural/functional homolog of the M-cluster (or cofactor; [(R-homocitrate)MoFe 7 S 9 C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster–bound NifDK to mimic its NifEN counterpart and enable N 2 reduction. These observations, coupled with phylogenetic, ecological, and mechanistic considerations, lead to the proposal of a NifEN-like, L-cluster–carrying protein as an ancient nitrogenase, the exploration of which could shed crucial light on the evolutionary origin of nitrogenase and related enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli

ABSTRACT The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii ( Av NifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active Av NifH protein upon co-expression of this protein with Av IscS/U and Av NifM in E. coli . Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed Av NifH protein has a high [Fe 4 S 4 ] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that Av NifM could serve as a chaperone and assist the maturation of a cluster-replete Av NifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the “clean” genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Heterologous synthesis of the complex homometallic cores of nitrogenase P- and M-clusters in Escherichia coli

Nitrogenase is an active target of heterologous expression because of its importance for areas related to agronomy, energy, and environment. One major hurdle for expressing an active Mo-nitrogenase in Escherichia coli is to generate the complex metalloclusters (P- and M-clusters) within this enzyme, which involves some highly unique bioinorganic chemistry/metalloenzyme biochemistry that is not generally dealt with in the heterologous expression of proteins via synthetic biology; in particular, the heterologous synthesis of the homometallic P-cluster ([Fe 8 S 7 ]) and M-cluster core (or L-cluster; [Fe 8 S 9 C]) on their respective protein scaffolds, which represents two crucial checkpoints along the biosynthetic pathway of a complete nitrogenase, has yet to be demonstrated by biochemical and spectroscopic analyses of purified metalloproteins. Here, we report the heterologous formation of a P-cluster-containing NifDK protein upon coexpression of Azotobacter vinelandii nifD, nifK, nifH, nifM, and nifZ genes, and that of an L-cluster-containing NifB protein upon coexpression of Methanosarcina acetivorans nifB, nifS, and nifU genes alongside the A. vinelandii fdxN gene, in E. coli. Our metal content, activity, EPR, and XAS/EXAFS data provide conclusive evidence for the successful synthesis of P- and L-clusters in a nondiazotrophic host, thereby highlighting the effectiveness of our metallocentric, divide-and-conquer approach that individually tackles the key events of nitrogenase biosynthesis prior to piecing them together into a complete pathway for the heterologous expression of nitrogenase. As such, this work paves the way for the transgenic expression of an active nitrogenase while providing an effective tool for further tackling the biosynthetic mechanism of this important metalloenzyme.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Incorporation of an Asymmetric Mo−Fe−S Cluster as an Artificial Cofactor into Nitrogenase

Abstract Nitrogenase employs a sophisticated electron transfer system and a Mo−Fe−S−C cofactor, designated the M‐cluster [(cit)MoFe 7 S 9 C]), to reduce atmospheric N 2 to bioaccessible NH 3 . 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 [Fe 6 S 9 (SEt) 2 ] 4− . Here, we demonstrate the utility of an asymmetric Mo−Fe−S cluster [Cp*MoFe 5 S 9 (SH)] 3− as an alternative artificial cofactor upon incorporation into the cofactor‐free nitrogenase scaffold. The resultant semi‐artificial enzyme catalytically reduces C 2 H 2 to C 2 H 4 , and CN − into short‐chain hydrocarbons, yet it is clearly distinct in activity from its [Fe 6 S 9 (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.

Tanifuji, Kazuki↗
DOE OSTI2022

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe 4 Se 4 ] Cluster

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 [Fe 4 Se 4 ] cluster that is used to substitute the [Fe4S4] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe 4 Se 4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe 4 Se 4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe 4 Se 4 ] 0 ) in the presence of dithionite, where its [Fe 4 S 4 ] counterpart in AvNifH exists solely in the reduced state ([Fe 4 S 4 ] 1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe 4 Se 4 ] and [Fe 4 S 4 ] 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.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Tracing the incorporation of the “ninth sulfur” into the nitrogenase cofactor precursor with selenite and tellurite

We report Molybdenum nitrogenase catalyses the reduction of N 2 to NH 3 at its cofactor, an [(R-homocitrate)MoFe 7 S 9 C] cluster synthesized via the formation of a [Fe 8 S 9 C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe 8 S 8 C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘ninth sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘ninth sulfur’ insertion remain elusive. Here we trace the ‘ninth sulfur’ insertion using SeO 3 2- and TeO 3 2- as ‘labelled’ SO 3 2- . Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the ‘ninth sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se 2- - and Te 2- -like species into the L-cluster. Density functional theory calculations further point to a plausible mechanism involving in situ reduction of SO 3 2- to S 2- , thereby suggesting the utility of this reaction to label the catalytically important belt region for mechanistic investigations of nitrogenase.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Characterization of a Mo-Nitrogenase Variant Containing a Citrate-Substituted Cofactor

Nitrogenase converts N 2 to NH 3 , and CO to hydrocarbons, at its cofactor site. In this work, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDK Cit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDK Cit reveals a shift of CO reduction from H 2 evolution toward hydrocarbon formation and an opposite shift of N 2 reduction from NH 3 formation toward H 2 evolution. Consistent with a shift in the Mo K-edge energy of NifDK Cit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structural evidence for a dynamic metallocofactor during N 2 reduction by Mo-nitrogenase

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N 2 ) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N 2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo–O5 (carboxyl) or Mo–O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process.

Science & Technology - Other Topics↗
DOE OSTI2020

Special Issue on Nitrogenases and Homologous Systems

The nitrogenase superfamily comprises homologous enzyme systems that carry out fundamentally important processes, including the reduction of N 2 and CO, and the biosynthesis of bacteriochlorophyll and coenzyme F430. This special issue provides a cross-disciplinary overview of the ongoing research in this highly diverse and unique research area of metalloprotein biochemistry.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗