External Electron Donor Research Articles

PsAA9A, a C1-specific AA9 lytic polysaccharide monooxygenase from the white-rot basidiomycete Pycnoporus sanguineus.

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus. KEY POINTS: • PsAA9A is the first AA9 from P. sanguineus to be characterized. • PsAA9A has activity on cellulose, producing C1-oxidized cello-oligosaccharides. • Boosting activity with GH1, GH5, and GH6 was proven.

Novel Integrated Biotrickling Filter-Anammox Bioreactor System for the Complete Treatment of Ammonia in Air with Nitrification and Denitrification.

An integrated biotrickling filter-anammox bioreactor system for the complete treatment of ammonia in air with conversion to nitrogen gas without the supply of an extraneous electron donor for denitrification was established. Partial nitritation (i.e., conversion of ammonium to nitrite) was successfully achieved in the biotrickling filter (BTF) through free ammonia (FA) and free nitrous acid (FNA) inhibition on nitrite-oxidizing bacteria (NOB). During transients, while increasing nitrogen loading, FA was the main inhibitor of ammonia-oxidizing bacteria (AOB) and NOB, while during a steady state, it was mainly FNA, which was responsible for inhibitory effects due to the accumulation of nitrite. Ammonia removal by the BTF reached 50 gN m-3 h-1 with 100% removal at an inlet concentration of 404 ppmv and a gas residence time of 21 s. Average removal of ammonia during stable operation was 95%. The anammox bioreactor was slightly undersized compared to the BTF and could remove 75% of total nitrogen discharged by the BTF when the two reactors were connected and liquid was in one-pass mode. This undersizing caused accumulation of nitrite in the system when liquid was circled in a quasi-closed loop, which gradually inhibited the activity of anammox bacteria. Overall, this study demonstrates that ammonia in air can be effectively treated and converted to harmless nitrogen gas without an external electron donor supply using a biotrickling filter combined with an anammox bioreactor.

Synthesis and properties of biomass eugenol-functionalized isotactic poly(1-butene)s

Functional isotactic poly(1-butene) (iPB) bearing natural antioxidant has been successfully synthesized via the MgCl2-supported TiCl4 Ziegler-Natta catalyst-mediated copolymerization of 1-butene with a renewable eugenol (EG) comonomer. The incorporated EG contents could be conveniently tailored by changing the comonomer loadings and reaction parameters such as temperature, molar ratio of co-catalyst/main catalyst (Ti), and external electron donor loading, up to 0.62 mol% EG units incorporated into iPB chain. Interestingly, the introduction of EG into the 1-butene polymerization are beneficial for improving the molecular weight of resultant copolymers. EG-functionalized iPB demonstrate a higher thermal-oxidative stability with respect to the pristine PB, nearly being proportional to the EG content, whose oxidation induction time (OIT) of iPB containing 0.62 mol% EG units is prolonged by 25 fold than that of iPB without EG units. Nevertheless, the EG counits in the iPB backbone limit the crystallization rate and suppress the Form II-Form I crystal transformation.

Humin Assists Reductive Acetogenesis in Absence of Other External Electron Donor.

The utilization of extracellular electron transfer by microorganism is highly engaging for remediation of toxic pollutants under “energy-starved” conditions. Humin, an organo-mineral complex of soil, has been instrumental as an external electron mediator for suitable electron donors in the remediative works of reductive dehalogenation, denitrification, and so forth. Here, we report, for the first time, that humin assists microbial acetogenesis as the extracellular electron donor using the electron acceptor . Humin was obtained from Kamajima paddy soil, Japan. The anaerobic acetogenic consortium in mineral medium containing as the inorganic carbon source used suspended humin as the energy source under mesophilic dark conditions. Retardation of acetogenesis under the -deficient conditions demonstrated that humin did not function as the organic carbon source but as electron donor in the -reducing acetogenesis. The consortium with humin also achieved anaerobic dechlorination with limited methanogenic activity. Total electron-donating capacity of humin was estimated at about 87 µeeq/g-humin. The metagenomic sequencing of 16S rRNA genes showed the predominance of Firmicutes (71.8 ± 2.5%) in the consortium, and Lachnospiraceae and Ruminococcaceae were considered as the -reducing acetogens in the consortium. Thus, microbial fixation of using humin introduces new insight to the holistic approach for sustainable treatment of contaminants in environment.

Assessment of 2,4-Dinitroanisole Transformation Using Compound-Specific Isotope Analysis after In Situ Chemical Reduction of Iron Oxides.

Ferrous iron-bearing minerals are important reductants in the contaminated subsurface, but their availability for the reduction of anthropogenic pollutants is often limited by competition with other electron acceptors including microorganisms and poor accessibility to Fe(II) in complex hydrogeologic settings. The supply of external electron donors through in situ chemical reduction (ISCR) has been proposed as one remediation approach, but the quantification of pollutant transformation is complicated by the perturbations introduced to the subsurface by ISCR. Here, we evaluate the application of compound specific isotope analysis (CSIA) for monitoring the reduction of 2,4-dinitroanisole (DNAN), a component of insensitive munitions formulations, by mineral-bound Fe(II) generated through ISCR of subsurface material from two field sites. Electron balances from laboratory experiments in batch and column reactors showed that 3.6% to 11% of the total Fe in the sediments was available for the reduction of DNAN and its partially reduced intermediates after dithionite treatment. The extent of DNAN reduction was successfully quantified from its N isotope fractionation measured in the column effluent based on the derivation of a N isotope enrichment factor, εN, derived from a comprehensive series of isotope fractionation experiments with numerous Fe(II)-bearing minerals as well as dithionite-reduced subsurface materials. Our observations illustrate the utility of CSIA as a robust approach to evaluate the success of in situ remediation through abiotic contaminant reduction.

Nitrate removal performance and diversity of active denitrifying bacteria in denitrification reactors using poly(L-lactic acid) with enhanced chemical hydrolyzability.

Poly(L-lactic acid) (PLLA) can be used as an external electron donor in denitrification reactors to treat drinking water, aquaculture water, and industrial wastewater with an imbalanced carbon/nitrogen ratio. However, for PLLA to function in these applications, its chemical hydrolyzability requires improvement. Although the adjustment of the crystallinity (Xc) is effective in improving the hydrolyzability of PLLA, the condition for the Xc of PLLA, in which a sufficient amount of lactic acid is released for denitrification, must be clarified. Therefore, this study investigated the effective Xc range and optimal PLLA content as an electron donor for continuous nitrate removal in denitrification reactors. This study also explored the abundance, succession, and diversity of active denitrifying bacteria in denitrification reactors. The nitrate removal activity of activated sludge using the highly crystalline PLLA (Xc = 39.4%) was 1.8 mg NO3- -N g MLSS-1 h-1, which is 2.4 times higher than that using the nearly amorphous PLLA (Xc = 0.9%). During the 57 days of operation, the denitrification reactor with 3% (w/v) highly crystalline PLLA continued to completely remove nitrate, with a maximum nitrate removal activity of 22.8 mg NO3- -N g MLSS-1 h-1. The 16S rRNA amplicon sequencing and clone library analyses are using transcripts of two nitrite reductase genes, encoding cytochrome cd1 nitrite reductase, and copper-containing nitrite reductase revealed that bacteria belonging to the families Comamonadaceae, Rhodocyclaceae, and Alcaligenaceae were active denitrifying bacteria in the denitrification reactor using PLLA.

The liquid fraction from hydrothermal pretreatment of wheat straw provides lytic polysaccharide monooxygenases with both electrons and H2O2 co-substrate

BackgroundEnzyme-aided valorization of lignocellulose represents a green and sustainable alternative to the traditional chemical industry. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important components of the state-of-the art enzyme cocktails for cellulose conversion. Yet, these monocopper enzymes are poorly characterized in terms of their kinetics, as exemplified by the growing evidence for that H2O2 may be a more efficient co-substrate for LPMOs than O2. LPMOs need external electron donors and one key question of relevance for bioprocess development is whether the required reducing power may be provided by the lignocellulosic substrate.ResultsHere, we show that the liquid fraction (LF) resulting from hydrothermal pretreatment of wheat straw supports LPMO activity on both chitin and cellulose. The initial, transient activity burst of the LPMO reaction was caused by the H2O2 present in the LF before addition of LPMO, while the steady-state rate of LPMO reaction was limited by the LPMO-independent production of H2O2 in the LF. H2O2 is an intermediate of LF oxidation as evidenced by a slow H2O2 accumulation in LF, despite high H2O2 production rates. This H2O2 scavenging ability of LF is important since high concentrations of H2O2 may lead to irreversible inactivation of LPMOs.ConclusionsOur results support the growing understanding that fine-tuned control over the rates of H2O2 production and consumption in different, enzymatic and non-enzymatic reactions is essential for harnessing the full catalytic potential of LPMOs in lignocellulose valorization.

Cr(VI) removal performance from aqueous solution by Pseudomonas sp. strain DC-B3 isolated from mine soil: characterization of both Cr(VI) bioreduction and total Cr biosorption processes.

Microbial methods are promising and environmentally friendly methods for remediating heavy metal contamination. In this study, a Cr(VI)-resistant bacterial strain, DC-B3, which was identified as Pseudomonas sp. by 16S rDNA gene sequencing, was isolated from heavy metal-contaminated mine soil, and its performance in Cr(VI) removal from wastewater in terms of Cr(VI) reduction and total Cr adsorption was assessed. This strain exhibited a high capability to reduce Cr(VI) to less toxic Cr(III) without the addition of an external electron donor at low pH (2.0). The Cr(VI) reduction capacity and rate both increased linearly with increasing Cr(VI) concentration, with a reduction capacity of 32.0 mg Cr(VI)·g-1 achieved at an initial concentration of 135.0 mg L-1 over 75 h. In addition, 41.0% of the total Cr was removed from the solution by biosorption, and equilibrium was reached within approximately 5 h. The total Cr sorption process was well described by the pseudo-second-order kinetic and Langmuir isotherm models. Desorption assays indicated that NaOH was the most efficient agent for total Cr desorption, and Cr(VI) and generated Cr(III) were both loaded on the DC-B3 biomass. The bacterial cells after Cr treatment were characterized by scanning electron microscopy-energy dispersive X-ray spectrometer and Fourier transform infrared spectroscopy analyses. Strain DC-B3 showed high potential for possible application in the remediation of Cr(VI) contamination in mine areas.

Key sub-community dynamics of medium-chain carboxylate production

BackgroundThe carboxylate platform is a promising technology for substituting petrochemicals in the provision of specific platform chemicals and liquid fuels. It includes the chain elongation process that exploits reverse β–oxidation to elongate short-chain fatty acids and forms the more valuable medium-chain variants. The pH value influences this process through multiple mechanisms and is central to effective product formation. Its influence on the microbiome dynamics was investigated during anaerobic fermentation of maize silage by combining flow cytometric short interval monitoring, cell sorting and 16S rRNA gene amplicon sequencing.ResultsCaproate and caprylate titres of up to 6.12 g L−1 and 1.83 g L−1, respectively, were achieved in a continuous stirred-tank reactor operated for 241 days. Caproate production was optimal at pH 5.5 and connected to lactate-based chain elongation, while caprylate production was optimal at pH 6.25 and linked to ethanol utilisation. Flow cytometry recorded 31 sub-communities with cell abundances varying over 89 time points. It revealed a highly dynamic community, whereas the sequencing analysis displayed a mostly unchanged core community. Eight key sub-communities were linked to caproate or caprylate production (rS > | ± 0.7|). Amongst other insights, sorting and subsequently sequencing these sub-communities revealed the central role of Bifidobacterium and Olsenella, two genera of lactic acid bacteria that drove chain elongation by providing additional lactate, serving as electron donor.ConclusionsHigh-titre medium-chain fatty acid production in a well-established reactor design is possible using complex substrate without the addition of external electron donors. This will greatly ease scaling and profitable implementation of the process. The pH value influenced the substrate utilisation and product spectrum by shaping the microbial community. Flow cytometric single cell analysis enabled fast, short interval analysis of this community and was coupled with 16S rRNA gene amplicon sequencing to reveal the major role of lactate-producing bacteria.

Improving Selectivity and Activity of CO2 Reduction Photocatalysts with Oxygen

Summary A highly porous photocatalyst (copper on TiO2 aerogel) was synthesized and applied in aqueous CO2 reduction without using external sacrificial electron donors. For the first time, complete selectivity toward CO and improved catalyst productivity are observed in the presence of oxygen. The optimal activity is achieved in a feed containing 0.5 vol% O2 in CO2. In situ XAS, EPR, and UV-vis measurements suggest, among different Cu oxidation states, Cu(I) to be the most active species in photocatalytic CO2 reduction. Oxygen sensing of the catalyst in the presence of O2/CO2 mixtures indicated an unexpected photoadsorption of oxygen on the titania surface. We propose photooxidation of surface hydroxyl groups to be the electron source for CO2 reduction, which is supported by hydroxyl group consumption, detection of hydroxyl radicals using in situ EPR, and detection of surface peroxide species after the reaction.

Biological perchlorate reduction: which electron donor we can choose?

Biological reduction is an effective method for removal of perchlorate (ClO4-), where perchlorate is transformed into chloride by perchlorate-reducing bacteria (PRB). An external electron donor is required for autotrophic and heterotrophic reduction of perchlorate. Therefore, plenty of suitable electron donors including organic (e.g., acetate, ethanol, carbohydrate, glycerol, methane) and inorganic (e.g., hydrogen, zero-valent iron, element sulfur, anthrahydroquinone) as well as the cathode have been used in biological reduction of perchlorate. This paper reviews the application of various electron donors in biological perchlorate reduction and their influences on treatment efficiency of perchlorate and biological activity of PRB. We discussed the criteria for selection of appropriate electron donor to provide a flexible strategy of electron donor choice for the bioremediation of perchlorate-contaminated water.

Nitrogen removal in response to plants harvesting in two kinds of enhanced hydroponic root mats treating secondary effluent

Hydroponic root mats (HRMs) are a green technology for various wastewaters purification. However, plants wilting will inevitably reduce the purification efficiency of HRMs. Harvesting as an important way of plant management for a better understanding of sustainability of HRMs has always been highly controversial. The goal of this study was to investigate the impacts and sustainability of harvesting on nitrogen removal and greenhouse gases (CH4, N2O) emissions of the two kinds of enhanced hydroponic root mats: autotrophic hydroponic root mat (AHRM) and heterotrophic hydroponic root mat (HHRM) for treating secondary effluent. The results showed that harvesting temporarily decreased nitrogen removal efficiency of the two systems, and removal efficiency recovered quickly because of the existence of external electron donors. The effects of harvesting are ordered as: HHRM > AHRM, NO3−-N > NH4+-N. Increasing C/N, S/N would reduce the impact of temperature on harvesting systems. Harvesting also increased the emission of greenhouse gases, and increasing C/N(=6), S/N(=1.1) could significantly reduce greenhouse gases emission of the harvesting systems at low temperature. In addition, composition analysis of the shoots of the harvested plants was also conducted, and the results showed that N contents of growing shoots were significantly higher than that of withered shoots. In order to make the hydroponic root mats sustainable, harvesting before the plants wilt is more effective in removing nitrogen from the system permanently and maintain a sustainable system.

Isolation of Novel CO Converting Microorganism Using Zero Valent Iron for a Bioelectrochemical System (BES)

Carbon monoxide (CO) is one of the main waste gas components of the steel industry and biomass gasification process. CO has also been highlighted as a feedstock for biological conversion to platform and valueadded chemicals. Conventional CO-converting strains have drawbacks of slow growth rate and high sensitivity to oxygen as well as low conversion yield. Most CO conversion microbes harbor the Wood-Ljungdahl pathway (WLP) and CO-dehydrogenase, and the reducing equivalent is significantly limited for acetyl-CoA synthesis. In this study, electrochemically active CO converting strains were isolated and characterized using zero valent iron (ZVI) granules (Fe0) as an external electron donor. The strains isolated from ZVI augmented enrichment could also use a carbon electrode as the electron donor, and simultaneously convert CO to acetate and VFAs in a bioelectrochemical system. From enrichment and isolation with ZVI, both Clostridium sp. HN02 and Fonticella sp. HN43 were isolated and showed higher performance for acetate production from CO in BES, and electrochemical activity by cyclic voltammetry.

Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2 When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a "priming reduction" reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (KmRapp). Theoretical analyses revealed that KmRapp decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.

Is microbial reduction of Fe (III) in podzolic soils influencing C release?

Fe(III) oxides stabilize soil organic matter by forming Fe organo-mineral associations (Fe-OMA). Under anoxic conditions, Fe-OMA may be destabilized by microbial dissimilatory iron reduction that releases aqueous Fe(II) and also possibly adsorbed or co-precipitated organic matter. Soil spodic horizons that accumulate Fe-OMA are ideal natural materials to study such impact. Here, we study three spodic horizons from pedons (P) of increasing age: P-270 yrs, P-330 yrs and P-530 yrs. Their contents of total carbon and short range ordered (SRO) Fe oxides increase with soil age from, respectively, 1486 to 3618 and 13 to 249 μmol g−1. The samples were incubated for 96 h under anoxic conditions, with and without Shewanella putrefaciens (a model dissimilatory Fe(III)-reducing bacteria) in a minimal medium devoid of any pH buffer and external electron donor. The concentration of dissolved Fe(II), total Fe and organic C was monitored at 9 time steps. With increasing age, both the rate and extent of microbial Fe(III) reduction increased after S. putrefaciens addition. In P-270 yrs, P-330 yrs and P-530 yrs, respectively, the microbial reduction rates were 0.004, 0.026 and 0.114 fmol Fe(II) h−1 cell−1 while the amounts of released Fe(II) were 0.23, 0.32 and 1.98 μmol Fe(II) g−1, both being strongly correlated with SRO Fe oxides content. Dissolved organic carbon (DOC) was released with or without S. putrefaciens in all samples (up to 73 μmol OC g−1 after 96 h in P-530 yrs). Adding S. putrefaciens significantly increased DOC release, but only in P-270 yrs. Podzol development thus increases the impact of anoxia on Fe(II) release since organic matter accumulation impedes Fe oxide crystallization, thereby amplifying Fe availability for Fe reducing microbes. The evolution of Fe oxide content and crystallinity thus affects the fate of both C and Fe in soils.

Exploring anammox bacteria ecology to improve nitrogen removal in wastewater treatment

Exploring anammox bacteria ecology to improve nitrogen removal in wastewater treatment

Solvent effect in 1,3-butadiene polymerization by cyclopentadienyl titanium trichloride (CpTiCl3)/methylaluminoxane (MAO) and pentamethylcyclopentadienyl titanium trichloride (Cp*TiCl3)/MAO catalysts

Stereospecific cis-1,4 polymerization of 1,3 butadiene is carried out by using CpTiCl3/MAO and Cp*TiCl3/MAO catalysts in solvents (mesitylene, toluene, benzene, chlorobenzene, m-chlorobenzene, hexafluorobenzene) having different nucleophilicity. The influence of nucleophilicity of used solvent on activity and selectivity of the two considered catalytic systems is evaluated. Catalyst activity is strongly affected by used solvent, while polymer microstructure remains almost unchanged. The formation of a labile specie anti-η3-π-butenyl-Ti coordinated to solvent molecule, able to increase the rate of polymerization reaction, is hypothesized. The effect of two different external electron donors, N(C2H5)3 and P(C6H5)3, in the polymerization environment on activity and selectivity of the two considered catalytic systems is also investigated. Both N(C2H5)3 and P(C6H5)3 induce a sweeping decrease of catalyst activity, but do not cause changes in polymer microstructures.

Synthesis of Perylene-Tagged Internal and External Electron Donors for Magnesium Dichloride Supported Ziegler–Natta Catalysts

We report on the synthesis of three perylene-tagged electron donors representing three major types – phthalates, diethers, and alkoxysilanes – which are of importance for the subsequent studies of MgCl2-supported Ziegler–Natta catalysts by means of laser scanning confocal fluorescence microscopy. The obtained products were unambiguously characterized, including by X-ray crystal structure analysis; their photophysical properties (absorption and emission spectra) were investigated as well. Additionally, a reliable and convenient protocol for the multigram synthesis of the required starting material – 3-bromo­perylene (PerBr) – was developed. The key step of this method was synthesis of trialkylsilyl-substituted perylenes, which were further separated by means of flash chromatography followed by conversion of the isolated 3-trialkylsilyl-substituted product to PerBr.

Ziegler-Natta Catalyst Based on MgCl₂/Clay/ID/TiCl₄ for the Synthesis of Spherical Particles of Polypropylene Nanocomposites.

In the present work, we have designed MgCl2/clay/internal donor (ID)/TiCl4 based bisupported Ziegler-Natta catalysts containing varying amounts of organoclay (montmorillonite) in order to synthesize spherical particles of polypropylene/clay nanocomposites (PCN). The organoclay was introduced into the catalyst support formulation and PCN was obtained using the in situ polymerization technique. Decreasing the reaction time, it was possible to obtain nanocomposites with high concentrations of clay (masterbatches). Micrographs of SEM confirmed the spherical morphology of the catalysts. In addition, XRD patterns show that the active sites for polymerization were inserted in the clay galleries. The catalytic performance was evaluated in slurry propylene polymerization using triethylaluminium as cocatalyst and silane as external electron donor at 70 °C, 4 bar, and different reaction times. The PCNs obtained containing different clay amounts were characterized by X-ray diffraction, thermal analyses, transmission electronic microscopy, and extractables in heptane. The results revealed that the synthesized PP/clay particles were also spherical showing that the morphological control is possible even using catalysts containing high amounts of clay. The PCN presented high degradation temperature (459 °C). The XRD peak related to the clay interlamellar distance has shifted to lower angles, and TEM images confirmed the formation of exfoliated/intercalated clay on the PP matrix and absence of microparticles of clay.

Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.