Nitrogenase Iron Protein Research Articles

Acidisoma cladoniae sp. nov., an acidotolerant bacterium isolated from an Antarctic lichen.

Gram-staining-negative, aerobic, white-cream-pearly colony, coccobacilli, and non-motile bacterial strain, PAMC 29798T was isolated from an Antarctic lichen. The strain was acidotolerant and psychrotolerant growing at pH 4.0-7.5 (optimally at pH 4.0-6.5) and 0-25°C (optimally at 10-20°C). The major fatty acids are Summed Feature 8, C18:1 2OH, and C19:0 cyclo ω8c. The major respiratory quinone was Q-10. Phylogenetic and phylogenomic analyses indicated that strain PAMC 29798T belonged to the genus Acidisoma and 16S rRNA gene sequences of PAMC 29798T were closely related to Acidisoma silvae (97.7% sequence similarity), Acidisoma cellulosilyticum (96.5%), Acidisoma tundrae (96.5%), and Acidisoma sibiricum (96.3%). Genomic relatedness analyses showed that strain PAMC 29798T was clearly distinguished from type strains of the genus Acidisoma based on values of average nucleotide identity (< 75%) and the digital DNA-DNA hybridization (< 19.6%). Genome analysis revealed that the genome size of PAMC 29798T is approximately 5.0 Mb with a G+C content of 63.4%. The complete genome comprises 5 contigs containing 4636 protein-coding genes, 46 tRNA genes, and 2 rRNA operons. The genome possesses genes for light-harvesting complexes, type-II photosynthetic reaction center, and C-P lyase to solubilize organic phosphates, while genes encoding nitrogenase iron protein involved in the nitrogen fixation were not present. Based on the results of phylogenetic, genome-based relatedness, and physiological and genomic analyses, strain PAMC 29798T is proposed to represent a novel species of the genus Acidisoma, with the name Acidisoma cladoniae. The type strain is PAMC 29798T (= KCTC 82159T = JCM 35634T).

Computational Structural and Functional Characterization of Azorhizobium caulinodans NifH Protein and Associated Cluster Analysis

Nitrogenase iron protein, a key enzyme in biological nitrogen fixation, is required for turning atmospheric nitrogen into a physiologically usable form. In the present work, nitrogenase iron protein (encoded by the nifH gene; WP_012169572.1) of Azorhizobium caulinodans, a nitrogen-fixing bacteria recognized for its symbiotic relationship with leguminous plants, was protein of interest. The nitrogenase iron proteins cluster with NifH were identified computationally and characterized using various tools.

Water layer and radiation damage effects on the orientation recovery of proteins in single-particle imaging at an X-ray free-electron laser

The noise caused by sample heterogeneity (including sample solvent) has been identified as one of the determinant factors for a successful X-ray single-particle imaging experiment. It influences both the radiation damage process that occurs during illumination as well as the scattering patterns captured by the detector. Here, we investigate the impact of water layer thickness and radiation damage on orientation recovery from diffraction patterns of the nitrogenase iron protein. Orientation recovery is a critical step for single-particle imaging. It enables to sort a set of diffraction patterns scattered by identical particles placed at unknown orientations and assemble them into a 3D reciprocal space volume. The recovery quality is characterized by a “disconcurrence” metric. Our results show that while a water layer mitigates protein damage, the noise generated by the scattering from it can introduce challenges for orientation recovery and is anticipated to cause problems in the phase retrieval process to extract the desired protein structure. Compared to these disadvantageous effects due to the thick water layer, the effects of radiation damage on the orientation recovery are relatively small. Therefore, minimizing the amount of residual sample solvent should be considered a crucial step in improving the fidelity and resolution of X-ray single-particle imaging experiments.

A complete biomimetic iron-sulfur cubane redox series

Synthetic iron-sulfur cubanes are models for biological cofactors, which are essential to delineate oxidation states in the more complex enzymatic systems. However, a complete series of [Fe4S4]n complexes spanning all redox states accessible by 1-electron transformations of the individual iron atoms (n = 0-4+) has never been prepared, deterring the methodical comparison of structure and spectroscopic signature. Here, we demonstrate that the use of a bulky arylthiolate ligand promoting the encapsulation of alkali-metal cations in the vicinity of the cubane enables the synthesis of such a series. Characterization by EPR, 57Fe Mössbauer spectroscopy, UV-visible electronic absorption, variable-temperature X-ray diffraction analysis, and cyclic voltammetry reveals key trends for the geometry of the Fe4S4 core as well as for the Mössbauer isomer shift, which both correlate systematically with oxidation state. Furthermore, we confirm the S = 4 electronic ground state of the most reduced member of the series, [Fe4S4]0, and provide electrochemical evidence that it is accessible within 0.82 V from the [Fe4S4]2+ state, highlighting its relevance as a mimic of the nitrogenase iron protein cluster.

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4Se4] Cluster

AbstractThe 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 [Fe4Se4] 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 [Fe4Se4] cluster to adopt the super‐reduced, all‐ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4Se4] cluster in AvNifH already assumes a partial all‐ferrous state ([Fe4Se4]0) in the presence of dithionite, where its [Fe4S4] counterpart in AvNifH exists solely in the reduced state ([Fe4S4]1+). Such a discrepancy in the redox properties of the AvNifH‐associated [Fe4Se4] and [Fe4S4] 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.

Characterization of a Nitrogenase Iron Protein Substituted with a Synthetic [Fe4 Se4 ] Cluster.

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe4 S4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe4 Se4 ] cluster that is used to substitute the [Fe4 S4 ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4 Se4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4 Se4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe4 Se4 ]0 ) in the presence of dithionite, where its [Fe4 S4 ] counterpart in AvNifH exists solely in the reduced state ([Fe4 S4 ]1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe4 Se4 ] and [Fe4 S4 ] 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.

Microbacterium sulfonylureivorans sp. nov., isolated from sulfonylurea herbicides degrading consortium.

A novel Gram-stain positive, aerobic, motile, rod-shaped bacterium, designated strain LAM7116T was isolated from a sulfonylurea herbicides degrading consortium enriched with birch forest soil from Xinjiang. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain LAM7116T was closely related to the members of the genus Microbacterium, with the highest similarity to Microbacterium flavescens DSM 20643T (98.48%) and Microbacterium kitamiense Kitami C2T (98.48%). Strain LAM7116T formed a distinct subclade with M. flavescens DSM 20643T within the genus Microbacterium in the 16S rRNA gene phylogenetic trees. The genomic DNA G + C content of LAM7116T was 69.9mol%. The digital DNA-DNA hybridization (dDDH) value between strain LAM7116T and M. flavescens DSM 20643T was 27.20%. The average nucleotide identity (ANI) value was 83.96% by comparing the draft genome sequences of strain LAM7116T and M. flavescens DSM 20643T. The major fatty acids were anteiso-C15:0, anteiso-C17:0, iso-C17:0, and iso-C16:0. The respiratory menaquinones of strain LAM7116T were MK-13 and MK-14. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, an unidentified lipid, and an unidentified glycolipid. The peptidoglycan contained the amino acids glycine, lysine, alanine, and glutamic acid. Based on the phenotypic characteristics and genotypic analyses, we consider that strain LAM7116T represents a novel species, for which the name Microbacterium sulfonylureivorans sp. nov. was proposed. The type strain is LAM7116T (= CGMCC 1.16620T = JCM 32823T). Strain LAM7116T secreted auxin IAA and grew well in Ashby nitrogen-free culture medium. Genomic results showed that strain LAM7116T carried the nitrogenase iron protein (nifU and nifR3) gene, which indicated that strain LAM7116T has the potential to fix nitrogen and promote plant growth. At same time, strain LAM7116T can degrade nicosulfuron (a kind of sulfonylurea herbicides) using glucose as carbon source. Microbacterium sulfonylureivorans sp. nov. LAM7116T is a potential candidate for the biofertilizers of organic agriculture areas, and may possess potential to be used in bioremediation of nicosulfuron-contaminated environments.

Effects of mowing dominant grasses on root exudation and soil nitrogen cycling in a natural sod culture apple orchard

We evaluated the effects of mowing dominant grasses on root exudation and soil nitrogen (N) cycling by assessing metabolomics analysis of root exudates, microbial metabolism, the abundance of N-cycling-related prokaryotes, and different forms of N concentrations in soil. The treatments included Polygonum aviculare L. mowing (T1), Digitaria sanguinalis (L.) Scop. mowing (T2), and no mowing as the controls (CK1 and CK2). The results showed that compared with the no mowing control (CK1 and CK2), T1 and T2 root exudates contained 223 (178 up-regulated, 45 down-regulated) and 183 (40 up-regulated, 143 down-regulated) differential metabolites, respectively. The average well colour development (AWCD) could reflect the microbial metabolic activity. The AWCD values of T1 were increased while that of T2 decreased on the 2&lt;sup&gt;nd&lt;/sup&gt; day after mowing. The variation in root exudates was the main reason for the change in soil AWCD values and carbon utilisation of T1 and T2 on the 2&lt;sup&gt;nd&lt;/sup&gt; day after mowing. Mowing increased soil microbial biomass N content significantly in the T1 and T2 topsoil. The NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;–&lt;/sup&gt;-N and NH&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;+&lt;/sup&gt;-N contents in the 0–10 cm soil increased on the 2&lt;sup&gt;nd&lt;/sup&gt; day after T1 mowing with an increase in the nitrogenase iron protein gene (nifH), glutamate dehydrogenase gene (gdh), ammonia monooxygenase gene (amoA) of ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) abundance. However, NO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;–&lt;/sup&gt;-N content decreased on the 2&lt;sup&gt;nd&lt;/sup&gt; day after T2 mowing following a decrease in AOA-amoA and AOB-amoA gene abundance. The results of this study will facilitate the optimisation of sod culture orchard N management, reduction of N fertiliser input, and improvement of N utilisation efficiency.

Vegetative cells may perform nitrogen fixation function under nitrogen deprivation in Anabaena sp. strain PCC 7120 based on genome-wide differential expression analysis.

Nitrogen assimilation is strictly regulated in cyanobacteria. In an inorganic nitrogen-deficient environment, some vegetative cells of the cyanobacterium Anabaena differentiate into heterocysts. We assessed the photosynthesis and nitrogen-fixing capacities of heterocysts and vegetative cells, respectively, at the transcriptome level. RNA extracted from nitrogen-replete vegetative cells (NVs), nitrogen-deprived vegetative cells (NDVs), and nitrogen-deprived heterocysts (NDHs) in Anabaena sp. strain PCC 7120 was evaluated by transcriptome sequencing. Paired comparisons of NVs vs. NDHs, NVs vs. NDVs, and NDVs vs. NDHs revealed 2,044 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the DEGs showed that carbon fixation in photosynthetic organisms and several nitrogen metabolism-related pathways were significantly enriched. Synthesis of Gvp (Gas vesicle synthesis protein gene) in NVs was blocked by nitrogen deprivation, which may cause Anabaena cells to sink and promote nitrogen fixation under anaerobic conditions; in contrast, heterocysts may perform photosynthesis under nitrogen deprivation conditions, whereas the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Immunofluorescence analysis of nitrogenase iron protein suggested that the nitrogen fixation capability of vegetative cells was promoted by nitrogen deprivation. Our findings provide insight into the molecular mechanisms underlying nitrogen fixation and photosynthesis in vegetative cells and heterocysts at the transcriptome level. This study provides a foundation for further functional verification of heterocyst growth, differentiation, and water bloom control.

Probing the All-Ferrous States of Methanogen Nitrogenase Iron Proteins.

The Fe protein of nitrogenase reduces two C1 substrates, CO2 and CO, under ambient conditions when its [Fe4S4] cluster adopts the all-ferrous [Fe4S4]0 state. Here, we show disparate reactivities of the nifH- and vnf-encoded Fe proteins from Methanosarcina acetivorans (designated MaNifH and MaVnfH) toward C1 substrates in the all-ferrous state, with the former capable of reducing both CO2 and CO to hydrocarbons, and the latter only capable of reducing CO to hydrocarbons at substantially reduced yields. EPR experiments conducted at varying solution potentials reveal that MaVnfH adopts the all-ferrous state at a more positive reduction potential than MaNifH, which could account for the weaker reactivity of the MaVnfH toward C1 substrates than MaNifH. More importantly, MaVnfH already displays the g = 16.4 parallel-mode EPR signal that is characteristic of the all-ferrous [Fe4S4]0 cluster at a reduction potential of −0.44 V, and the signal reaches 50% maximum intensity at a reduction potential of −0.59 V, suggesting the possibility of this Fe protein to access the all-ferrous [Fe4S4]0 state under physiological conditions. These results bear significant relevance to the long-lasting debate of whether the Fe protein can utilize the [Fe4S4]0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes.

Bacillus telluris sp. nov. Isolated from Greenhouse Soil in Beijing, China.

A novel Gram-stain-positive, rod-shaped, endospore-forming bacterium, which we designated as strain 03113T, was isolated from greenhouse soil in Beijing, China. Phylogenetic analysis based on 16S rRNA gene sequences showed strain 03113T is in the genus Bacillus and had the highest similarity to Bacillus solani CCTCC AB 2014277T (98.14%). The strain grew at 4 °C–50 °C (optimum 37 °C), with 0–10% (w/v) NaCl (optimum 5%), and in the range of pH 3.0–12.0 (optimum pH 8.0). Menaquinone was identified as MK-7, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The main major cellular fatty acids detected were anteiso-C15:0 (51.35%) and iso-C15:0 (11.06%), which are the predominant cellular fatty acids found in all recognized members of the genus Bacillus. The 16S rRNA gene sequence and core-genome analysis, the average nucleotide identity (ANI), and in silico DNA—DNA hybridization (DDH) value between strain 03113T and the most closely related species were 70.5% and 22.6%, respectively, which supported our conclusion that 03113T represented a novel species in the genus Bacillus. We demonstrated that type strain 03113T (=ACCC 03113T=JCM 33017T) was a novel species in the genus Bacillus, and the name Bacillus telluris sp. nov. was proposed. Strain 03113T secreted auxin IAA and carried the nitrogenase iron protein (nifH) gene, which indicated that strain 03113T has the potential to fix nitrogen and promote plant growth. Bacillus telluris sp. nov. 03113T is a potential candidate for the biofertilizers of organic agriculture areas.

Un-culturable Microbial Community Analysis of Paddy Ecosystem at Different Depths using Nif H Gene DGGE Analysis Approach

Background: Biological N2 fixation is gaining importance in rice ecosystem, because of current concern on the environmental and soil health that are caused by the continuous use of nitrogenous fertilizers and the need for improved sustainable rice productivity. BNF is the conversion of atmospheric nitrogen by certain microorganism’s viz., N2 fixing microorganisms or diazotrophs into forms that plants can use. A substantial molecular diversity of N2 fixing bacteria has been detected in the rice field soil based on retrieval of nif H gene fragment from DNA by molecular approach, i.e., DGGE (Denaturant Gradient Gel Electrophoresis). The present investigation aimed to assess the diversity of N2 fixing bacteria by sequencing and phylogenetic analysis of nif H gene of the metagenomic DNA collected from different depth of flooded rice soil. Methods: In this laboratory investigation, soil samples were collected from the paddy field at different depths viz., rhizosphere region, 5 cm, 10 cm, 15 cm depth using the core sampler during active tillering stage. In the laboratory, the collected soil samples were analyzed by molecular methods involving the use of primer sets based on nif H sequences (nitrogenase reductase). Result: Our investigations in the genetic diversity of diazotrophs associated with rice field soil using nif H revealed the existence of seven discrete bands. The sequencing results showed all sequences were classified as uncultured bacterium clone nitrogenase iron protein (nif H) gene, partial cds. The present works based on the applications of molecular methods have facilitated the study of individual ecosystem and their specific activity.

Frontispiece: Structural and Mechanistic Insights into CO2 Activation by Nitrogenase Iron Protein

Frontispiece: Structural and Mechanistic Insights into CO₂ Activation by Nitrogenase Iron Protein

Structural and Mechanistic Insights into CO2 Activation by Nitrogenase Iron Protein.

The Fe protein of nitrogenase catalyzes the ambient reduction of CO2 when its cluster is present in the all-ferrous, [Fe4 S4 ]0 oxidation state. Here, we report a combined structural and theoretical study that probes the unique reactivity of the all-ferrous Fe protein toward CO2 . Structural comparisons of the Azotobacter vinelandii Fe protein in the [Fe4 S4 ]0 and [Fe4 S4 ]+ states point to a possible asymmetric functionality of a highly conserved Arg pair in CO2 binding and reduction. Density functional theory (DFT) calculations provide further support for the asymmetric coordination of O by the "proximal" Arg and binding of C to a unique Fe atom of the all-ferrous cluster, followed by donation of protons by the proximate guanidinium group of Arg that eventually results in the scission of a C-O bond. These results provide important mechanistic and structural insights into CO2 activation by a surface-exposed, scaffold-held [Fe4 S4 ] cluster.

Enzymatic Reactions Involving Ketyls: From a Chemical Curiosity to a General Biochemical Mechanism.

Ketyls are radical anions with nucleophilic properties. Ketyls obtained by enzymatic one-electron reduction of thioesters were proposed as intermediates for the dehydration of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA. This concept was extended to the Birch-like reduction of benzoyl-CoA to 1,5-cyclohexadienecarboxyl-CoA. Nature uses two methods to achieve the therefore required low reduction potentials of less than -600 mV, either by an ATP-driven electron transfer similar to that catalyzed by the iron protein of nitrogenase or by electron bifurcation. Ketyls formed by thiyl radical-initiated oxidation of alcohols followed by deprotonation are involved in coenzyme B12-independent diol dehydratases, other glycyl radical enzymes mediating key reactions in the degradations of choline, taurine, and 4-hydroxyproline, and all three classes of ribonucleotide reductases. A special case is the dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which most likely proceeds via an oxidation to an allylic ketyl but requires neither a strong reductant nor an external radical generator.

Site-Specific Oxidation State Assignments of the Iron Atoms in the [4Fe:4S]2+/1+/0 States of the Nitrogenase Fe-Protein.

The nitrogenase iron protein (Fe‐protein) contains an unusual [4Fe:4S] iron‐sulphur cluster that is stable in three oxidation states: 2+, 1+, and 0. Here, we use spatially resolved anomalous dispersion (SpReAD) refinement to determine oxidation assignments for the individual irons for each state. Additionally, we report the 1.13‐Å resolution structure for the ADP bound Fe‐protein, the highest resolution Fe‐protein structure presently determined. In the dithionite‐reduced [4Fe:4S]1+ state, our analysis identifies a solvent exposed, delocalized Fe2.5+ pair and a buried Fe2+ pair. We propose that ATP binding by the Fe‐protein promotes an internal redox rearrangement such that the solvent‐exposed Fe pair becomes reduced, thereby facilitating electron transfer to the nitrogenase molybdenum iron‐protein. In the [4Fe:4S]0 and [4Fe:4S]2+ states, the SpReAD analysis supports oxidation states assignments for all irons in these clusters of Fe2+ and valence delocalized Fe2.5+, respectively.

Cluster-Dependent Charge-Transfer Dynamics in Iron-Sulfur Proteins.

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.

Activation and reduction of carbon dioxide by nitrogenase iron proteins.

The iron (Fe) proteins of molybdenum (Mo) and vanadium (V) nitrogenases mimic carbon monoxide (CO) dehydrogenase in catalyzing the interconversion between CO2 and CO under ambient conditions. Catalytic reduction of CO2 to CO is achieved in vitro and in vivo upon redox changes of the Fe-protein-associated [Fe4S4] clusters. These observations establish the Fe protein as a model for investigation of CO2 activation while suggesting its biotechnological adaptability for recycling the greenhouse gas into useful products.

High rates of microbial dinitrogen fixation and sulfate reduction associated with the Mediterranean seagrass Posidonia oceanica

Seagrass meadows of Posidonia oceanica represent hotspots of productivity in the oligotrophic Mediterranean Sea. The lack of dissolved inorganic nitrogen (DIN) in the seawater suggests that the N-demand of these meadows might be in part supported by microbial dinitrogen (N2) fixation. However, currently there are no direct N2 fixation measurements available for this productive marine macrophyte. Here we investigated N2 fixation activity associated with P. oceanica leaf, rhizome and root pieces. In 15N2 incubations, the roots exhibited highest rates of N2 fixation. The rates varied considerably between replicates, presumably due to a patchy microbial colonization of the roots. Additions of organic carbon compounds (acetate, glucose, sucrose or algal lysate) did not enhance the N2 fixation rates. Sulfate reduction rates measured alongside were also highest in root incubations. Correspondingly, sequences of the nifH gene (a marker gene for the iron protein of the N2-fixing enzyme nitrogenase) related to known sulfate-reducing bacteria were retrieved from P. oceanica roots. Other nifH sequences clustered with known heterotrophic diazotrophs previously identified in other marine macrophytes. In particular, many sequences obtained from P. oceanica roots were similar (>94%) to a saltmarsh rhizosphere-associated heterotrophic diazotroph, indicating that heterotrophic lifestyle might be common among marine macrophyte-associated diazotrophs.

X-ray Absorption and Emission Spectroscopic Studies of [L2Fe2S2](n) Model Complexes: Implications for the Experimental Evaluation of Redox States in Iron-Sulfur Clusters.

Herein, a systematic study of [L2Fe2S2]n model complexes (where L = bis(benzimidazolato) and n = 2-, 3-, 4-) has been carried out using iron and sulfur K-edge X-ray absorption (XAS) and iron Kβ and valence-to-core X-ray emission spectroscopies (XES). These data are used as a test set to evaluate the relative strengths and weaknesses of X-ray core level spectroscopies in assessing redox changes in iron–sulfur clusters. The results are correlated to density functional theory (DFT) calculations of the spectra in order to further support the quantitative information that can be extracted from the experimental data. It is demonstrated that due to canceling effects of covalency and spin state, the information that can be extracted from Fe Kβ XES mainlines is limited. However, a careful analysis of the Fe K-edge XAS data shows that localized valence vs delocalized valence species may be differentiated on the basis of the pre-edge and K-edge energies. These findings are then applied to existing literature Fe K-edge XAS data on the iron protein, P-cluster, and FeMoco sites of nitrogenase. The ability to assess the extent of delocalization in the iron protein vs the P-cluster is highlighted. In addition, possible charge states for FeMoco on the basis of Fe K-edge XAS data are discussed. This study provides an important reference for future X-ray spectroscopic studies of iron–sulfur clusters.