Search

Cytochrome c nitrite reductase, NrfA, is a soluble, periplasmic pentaheme cytochrome responsible for the reduction of nitrite to ammonium in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway, a vital reaction in the global nitrogen cycle. NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we summarize the latest progress in elucidating the reaction mechanism of ammonia production, including new findings about the active site architecture of NrfA, as well as recent results that elucidate electron transfer and storage in the pentaheme scaffold of this enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heme o is an Fe-porphyrin involved in the majority of aerobic respiration pathways found in all three domains of life. In eukaryotes and most aerobic prokaryotes, heme o functions solely as the precursor for the synthesis of heme a, a necessary cofactor for most heme-copper terminal oxidases. In some prokaryotes, such as Escherichia coli (E. coli), heme o can serve as a cofactor for heme-copper oxidases instead of heme a. Given its role as a key substrate or cofactor, purified heme o promises to be a valuable resource for the study of heme-copper oxidase assembly and activity. However, commercially available heme o is sold in limited quantities at a relatively high cost (compared to the prototypical heme b), making the use of heme o purchased from suppliers unfeasible for such studies. In this chapter, we present step-by-step methods both for heme o isolation from E. coli overexpressing heme o synthase and for HPLC analysis of cellular hemes (i.e., heme o and heme b)., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa 3 -type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site. Previous reports indicate that the C-terminal site is occupied by a heme b cofactor. The N-terminal site may function as the substrate (heme o) binding site, although this has not been confirmed experimentally. Here, we assess the role of conserved residues from the N- and C-terminal heme-binding sites in HAS from prokaryotic (Shewanella oneidensis) and eukaryotic (Saccharomyces cerevisiae) species - SoHAS/CtaA and ScHAS/Cox15, respectively. A glutamate within the N-terminal site is found to be critical for activity in both types of HAS, consistent with the hypothesis that a carbocation forms transiently during catalysis. In contrast, the residue occupying the analogous C-terminal position is dispensable for enzyme activity. In SoHAS, the C-terminal heme ligands are critical for stability, while in ScHAS, substitutions in either heme-binding site have little effect on global structure. In both species, in vivo accumulation of heme o requires the presence of an inactive HAS variant, highlighting a potential regulatory role for HAS in heme o biosynthesis., Competing Interests: Declarations of competing interest Declarations of interest: none., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: A lignocellulose-to-biofuel biorefinery process that enables multiple product streams is recognized as a promising strategy to improve the economics of this biorefinery and to accelerate technology commercialization. We recently identified an innovative pretreatment technology that enables of the production of sugars at high yields while simultaneously generating a high-quality lignin stream that has been demonstrated as both a promising renewable polyol replacement for polyurethane applications and is highly susceptible to depolymerization into monomers. This technology comprises a two-stage pretreatment approach that includes an alkaline pre-extraction followed by a metal-catalyzed alkaline-oxidative pretreatment. Our recent work demonstrated that H 2 O 2 and O 2 act synergistically as co-oxidants during the alkaline-oxidative pretreatment and could significantly reduce the pretreatment chemical input while maintaining high sugar yields (~ 95% glucose and ~ 100% xylose of initial sugar composition), high lignin yields (~ 75% of initial lignin), and improvements in lignin usage., Results: This study considers the economic impact of these advances and provides strategies that could lead to additional economic improvements for future commercialization. The results of the technoeconomic analysis (TEA) demonstrated that adding O 2 as a co-oxidant at 50 psig for the alkaline-oxidative pretreatment and reducing the raw material input reduced the minimum fuel selling price from $1.08/L to $0.85/L, assuming recoverable lignin is used as a polyol replacement. If additional lignin can be recovered and sold as more valuable monomers, the minimum fuel selling price (MFSP) can be further reduced to $0.73/L., Conclusions: The present work demonstrated that high sugar and lignin yields combined with low raw material inputs and increasing the value of lignin could greatly increase the economic viability of a poplar-based biorefinery. Continued research on integrating sugar production with lignin valorization is thus warranted to confirm this economic potential as the technology matures., (© 2022. The Author(s).)

Diaryl ethers undergo electrocatalytic hydrogenolysis (ECH) over skeletal Ni cathodes in a mild, aqueous process that achieves direct C-O cleavage without initial benzene ring saturation. Mechanistic studies find that aryl phenyl ethers with a single para or meta functional group (methyl, methoxy, or hydroxy) are selectively cleaved to the substituted benzene and phenol, in contrast to recently reported homogeneous catalytic cleavage processes. Ortho positioning of substituents reverses this C-O bond selectivity, except for the 2-phenoxyphenol case. Together with isotope labeling and co-solvent studies, these results point to two distinct cleavage mechanisms: (a) dual-ring coordination and C-H activation, leading to vicinal elimination to form phenol and a surface-bound aryne intermediate which is then hydrogenated and released as the arene; and (b) surface binding in keto form by the phenolic ring of the hydroxy-substituted substrates, followed by direct displacement of the departing phenol. Notably, acetone inhibits the well-known reduction of phenol to cyclohexanol, affording control of product ring saturation. A byproduct of this work is the discovery that the ECH treatment completely defluorinates substrates bearing aromatic C-F and C-CF 3 groupings., (© 2022. The Author(s).)

Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases contain a prenylated heme (typically heme a or occasionally heme o ) in the active site. In addition, many heme-copper oxidases, including mitochondrial cytochrome c oxidases, possess a second heme a cofactor. Despite the critical role of heme a in the electron transport chain, the details of the mechanism by which heme b , the prototypical cellular heme, is converted to heme o and then to heme a remain poorly understood. Recent structural investigations, however, have helped clarify some elements of heme a biosynthesis. In this review, we discuss the insight gained from these advances. In particular, we present a new structural model of heme o synthase (HOS) based on distance restraints from inferred coevolutionary relationships and refined by molecular dynamics simulations that are in good agreement with the experimentally determined structures of HOS homologs. We also analyze the two structures of heme a synthase (HAS) that have recently been solved by other groups. For both HOS and HAS, we discuss the proposed catalytic mechanisms and highlight how new insights into the heme-binding site locations shed light on previously obtained biochemical data. Finally, we explore the implications of the new structural data in the broader context of heme trafficking in the heme a biosynthetic pathway and heme-copper oxidase assembly.

Lignin has long been recognized as a potential feedstock for aromatic molecules; however, most lignin depolymerization methods create a complex mixture of products. The present study describes an alkaline aerobic oxidation method that converts lignin extracted from poplar into a collection of oxygenated aromatics, including valuable commercial compounds such as vanillin and p -hydroxybenzoic acid. Centrifugal partition chromatography (CPC) is shown to be an effective method to isolate the individual compounds from the complex product mixture. The liquid-liquid extraction method proceeds in two stages. The crude depolymerization mixture is first subjected to ascending-mode extraction with the Arizona solvent system L (pentane/ethyl acetate/methanol/water 2:3:2:3), enabling isolation of vanillin, syringic acid, and oligomers. The remaining components, syringaldehyde, vanillic acid, and p -hydroxybenzoic acid ( p HBA), were resolved by using ascending-mode extraction with solvent mixture comprising dichloromethane/methanol/water (10:6:4) separation. These results showcase CPC as an effective technology that could provide scalable access to valuable chemicals from lignin and other biomass-derived feedstocks., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Cytochrome c nitrite reductases (C c NIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

Lignin may serve as a renewable feedstock for the production of chemicals and fuels if mild, scalable processes for its depolymerization can be devised. The use of small organic thiols represents a bioinspired strategy to cleave the β-O-4 bond, the most common linkage in lignin. In the present study, synthetic β-O-4 linked polymers were treated with organic thiols, yielding up to 90 % cleaved monomer products. Lignin extracted from poplar was also treated with organic thiols resulting in molecular weight reductions as high as 65 % (M n ) in oxidized lignin. Thiol-based cleavage of other lignin linkages was also explored in small-molecule model systems to uncover additional potential pathways by which thiols might depolymerize lignin. The success of thiol-mediated cleavage on model dimers, polymers, and biomass-derived lignin illustrates the potential utility of small redox-active molecules to penetrate complex polymer matrices for depolymerization and subsequent valorization of lignin into fuels and chemicals., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca 2+ -independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca 2+ -dependent enzymes, suggesting convergent evolution., Competing Interests: Conflict of interest—The authors declare no conflicts of interest in regards to this work., (© 2020 Campeciño et al.)

We present here detailed mechanistic studies of electrocatalytic hydrogenation (ECH) in aqueous solution over skeletal nickel cathodes to probe the various paths of reductive catalytic C-O bond cleavage among functionalized aryl ethers relevant to energy science. Heterogeneous catalytic hydrogenolysis of aryl ethers is important both in hydrodeoxygenation of fossil fuels and in upgrading of lignin from biomass. The presence or absence of simple functionalities such as carbonyl, hydroxyl, methyl, or methoxyl groups is known to cause dramatic shifts in reactivity and cleavage selectivity between sp 3 C-O and sp 2 C-O bonds. Specifically, reported hydrogenolysis studies with Ni and other catalysts have hinted at different cleavage mechanisms for the C-O ether bonds in α-keto and α-hydroxy β-O-4 type aryl ether linkages of lignin. Our new rate, selectivity, and isotopic labeling results from ECH reactions confirm that these aryl ethers undergo C-O cleavage via distinct paths. For the simple 2-phenoxy-1-phenylethane or its alcohol congener, 2-phenoxy-1-phenylethanol, the benzylic site is activated via Ni C-H insertion, followed by beta elimination of the phenoxide leaving group. But in the case of the ketone, 2-phenoxyacetophenone, the polarized carbonyl π system apparently binds directly with the electron rich Ni cathode surface without breaking the aromaticity of the neighboring phenyl ring, leading to rapid cleavage. Substituent steric and electronic perturbations across a broad range of β-O-4 type ethers create a hierarchy of cleavage rates that supports these mechanistic ideas while offering guidance to allow rational design of the catalytic method. On the basis of the new insights, the usage of cosolvent acetone is shown to enable control of product selectivity.

Lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent oxidative enzymes, boost the degradation of polysaccharides such as cellulose, chitin, and others. While experimental methods are used to validate LPMO function, a computational method that can aid experimental methods and provide fast and accurate classification of sequences into LPMOs and its families would be an important step towards understanding the breadth of contributions these enzymes make in deconstruction of recalcitrant polysaccharides. In this study, we developed a machine learning-based tool called PreDSLpmo that employs two different approaches to functionally classify protein sequences into the major LPMO families (AA9 and AA10). The first approach uses a traditional neural network or multilayer percerptron-based approach, while the second employs bi-directional long short-term memory for sequence classification. Our method shows improvement in predictive power when compared with dbCAN2, an existing HMM-profile-based CAZyme predicting tool, on both validation and independent benchmark set., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The nucleophilic and reductive properties of thiolates and thiols make them ideal candidates as redox mediators via the thiol/disulfide couple. One mechanism for biological lignin depolymerization entails reduction of keto aryl ether bonds by an S N 2 mechanism with the thiol redox mediator glutathione. In this study, mimicking this chemistry in a simple protein- and metal-free process, several small organic thiols are surveyed for their ability to cleave aryl keto ethers that model the β-O-4 linkages found in partially oxidized lignin. In polar aprotic solvents, β-mercaptoethanol and dithiothreitol yielded up to 100 % formation of phenol and acetophenone products from 2-phenoxyacetophenone, but not from its reduced alcohol congener. The effects of reaction conditions and of substituents on the aryl rings and the keto ether linkage are assessed. These results, together with activation barriers computed by quantum chemical simulations and direct observation of the expected intermediate thioether, point to an S N 2 mechanism. This study confirms that small organic thiols can reductively break down lignin-relevant keto aryl ether linkages., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: In this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading. Lignins recovered from the pretreatments were characterized for polysaccharide content, molar mass distributions, β-aryl ether content, and response to depolymerization by thioacidolysis., Results: All three pretreatments resulted in significant solubilization of lignin and xylan, with the CELF pretreatment solubilizing the majority of both biopolymer categories. Enzymatic hydrolysis yields were shown to exhibit a strong, positive correlation with the lignin solubilized for the low enzyme loadings. The pretreatment-derived solubles in the [Ch][Lys]-pretreated biomass were presumed to contribute to inhibition of enzymatic hydrolysis in the eucalyptus as a substantial fraction of the pretreatment liquor was carried forward into hydrolysis for this pretreatment. The pretreatment-solubilized lignins exhibited significant differences in polysaccharide content, molar mass distributions, aromatic monomer yield by thioacidolysis, and β-aryl ether content. Key trends include a substantially higher polysaccharide content in the lignins recovered from the [Ch][Lys] pretreatment and high β-aryl ether contents and aromatic monomer yields from the Cu-AHP pretreatment. For all lignins, the 13 C NMR-determined β-aryl ether content was shown to be correlated with the monomer yield with a second-order functionality., Conclusions: Overall, it was demonstrated that the three pretreatments highlighted in this study demonstrated uniquely different functionalities in reducing biomass recalcitrance and achieving higher enzymatic hydrolysis yields for the hybrid poplar while yielding a lignin-rich stream that may be suitable for valorization. Furthermore, modification of lignin during pretreatment, particularly cleavage of β-aryl ether bonds, is shown to be detrimental to subsequent depolymerization., Competing Interests: Competing interestsThe authors declare the following competing financial interests: DBH, ELH, AB, NB (Methods of Using Multi-Ligand Metal Complexes and to Perform Oxidative Catalytic Pretreatment of Lignocellulosic Biomass—2019/0091674 A1). As a holder of this patent, we may benefit financially from advances in the technology discussed in this manuscript.

The heme a molecule is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. Heme a is synthesized from heme o by a multi-spanning inner membrane protein, heme a synthase (Cox15 in the yeast Saccharomyces cerevisiae ). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. To improve our understanding of heme a insertion into cytochrome c oxidase, here we investigated the protein-protein interactions that involve Cox15 in S. cerevisiae In addition to observing Cox15 in homooligomeric complexes, we found that a portion of Cox15 also associates with the mitochondrial respiratory supercomplexes. When supercomplex formation was abolished, as in the case of stalled cytochrome bc 1 or cytochrome c oxidase assembly, Cox15 maintained an interaction with select proteins from both respiratory complexes. In the case of stalled cytochrome bc 1 assembly, Cox15 interacted with the late-assembling cytochrome c oxidase subunit, Cox13. When cytochrome c oxidase assembly was stalled, Cox15 unexpectedly maintained its interaction with the cytochrome bc 1 protein, Cor1. Our results indicate that Cox15 and Cor1 continue to interact in the cytochrome bc 1 dimer even in the absence of supercomplexes or when the supercomplexes are destabilized. These findings reveal that Cox15 not only associates with respiratory supercomplexes, but also interacts with the cytochrome bc 1 dimer even in the absence of cytochrome c oxidase., (© 2018 Herwaldt et al.)

To improve sugar recovery and ethanol production from wheat straw, a sequential two-stage pretreatment process combining alkaline pre-extraction and acid catalyzed steam treatment was investigated. The results showed that alkaline pre-extraction using 8% (w/w) sodium hydroxide at 80 °C for 90 min followed by steam pretreatment with 3% (w/w) sulfur dioxide at 151 °C for 16 min was sufficient to prepare a substrate that could be efficiently hydrolyzed at high solid loadings. Moreover, alkaline pre-extraction reduced the process severity of steam pretreatment and decreased the generation of inhibitory compounds. During enzymatic hydrolysis, increasing solid loading decreased the yield of monomeric sugars. Enzymatic hydrolysis at 25% (w/v) solid loading, the yields of approximately 80% of glucose and 65% of xylose could be reached with an enzyme dosage of 25 mg protein/g glucan. Following fermentation of hydrolysate with sugar concentration of approximately 120 g/L, an ethanol concentration of 54.5 g/L was achieved., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and H 2 O 2 ) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss in sugar yield. We also performed technoeconomic analysis to determine if ethanol or water was the more cost-effective solvent during alkaline pre-extraction and if the expense associated with increasing the temperature was economically justified., Results: After Cu-AHP pretreatment of 120 °C NaOH-H 2 O pre-extracted and 120 °C NaOH-EtOH pre-extracted biomass, approximately 1.4-fold more total lignin was solubilized (78% and 74%, respectively) compared to the 30 °C NaOH-H 2 O pre-extraction (55%) carried out in a previous study. Consequently, increasing the temperature of the alkaline pre-extraction step to 120 °C in both ethanol and water allowed us to decrease bipyridine and H 2 O 2 during Cu-AHP and enzymes during hydrolysis with only a small reduction in sugar yields compared to 30 °C alkaline pre-extraction. Technoeconomic analysis indicated that 120 °C NaOH-H 2 O pre-extraction has the lowest installed ($246 million) and raw material ($175 million) costs compared to the other process configurations., Conclusions: We found that by increasing the temperature of the alkaline pre-extraction step, we could successfully lower the inputs for pretreatment and enzymatic hydrolysis. Based on sugar yields as well as capital, feedstock, and operating costs, 120 °C NaOH-H 2 O pre-extraction was superior to both 120 °C NaOH-EtOH and 30 °C NaOH-H 2 O pre-extraction.

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.

Background: Strategies to improve copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment of hybrid poplar were investigated. These improvements included a combination of increasing hydrolysis yields, while simultaneously decreasing process inputs through (i) more efficient utilization of H2O2 and (ii) the addition of an alkaline extraction step prior to the metal-catalyzed AHP pretreatment. We hypothesized that utilizing this improved process could substantially lower the chemical inputs needed during pretreatment., Results: Hybrid poplar was pretreated utilizing a modified process in which an alkaline extraction step was incorporated prior to the Cu-AHP treatment step and H2O2 was added batch-wise over the course of 10 h. Our results revealed that the alkaline pre-extraction step improved both lignin and xylan solubilization, which ultimately led to improved glucose (86 %) and xylose (95 %) yields following enzymatic hydrolysis. An increase in the lignin solubilization was also observed with fed-batch H2O2 addition relative to batch-only addition, which again resulted in increased glucose and xylose yields (77 and 93 % versus 63 and 74 %, respectively). Importantly, combining these strategies led to significantly improved sugar yields (96 % glucose and 94 % xylose) following enzymatic hydrolysis. In addition, we found that we could substantially lower the chemical inputs (enzyme, H2O2, and catalyst), while still maintaining high product yields utilizing the improved Cu-AHP process. This pretreatment also provided a relatively pure lignin stream consisting of ≥90 % Klason lignin and only 3 % xylan and 2 % ash following precipitation. Two-dimensional heteronuclear single-quantum coherence (2D HSQC) NMR and size-exclusion chromatography demonstrated that the solubilized lignin was high molecular weight (Mw ≈ 22,000 Da) and only slightly oxidized relative to lignin from untreated poplar., Conclusions: This study demonstrated that the fed-batch, two-stage Cu-AHP pretreatment process was effective in pretreating hybrid poplar for its conversion into fermentable sugars. Results showed sugar yields near the theoretical maximum were achieved from enzymatically hydrolyzed hybrid poplar by incorporating an alkaline extraction step prior to pretreatment and by efficiently utilizing H2O2 during the Cu-AHP process. Significantly, this study reports high sugar yields from woody biomass treated with an AHP pretreatment under mild reaction conditions.

Rationale: Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling., Methods: We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases., Results: All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.27–0.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.41–0.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases., Conclusions: The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.

Background: Alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2'-bipyridine complexes has previously been determined to substantially improve the enzymatic hydrolysis of woody plants including hybrid poplar as a consequence of moderate delignification. In the present work, cell wall morphological and lignin structural changes were characterized for this pretreatment approach to gain insights into pretreatment outcomes and, specifically, to identify the extent and nature of lignin modification., Results: Through TEM imaging, this catalytic oxidation process was shown to disrupt cell wall layers in hybrid poplar. Cu-containing nanoparticles, primarily in the Cu(I) oxidation state, co-localized with the disrupted regions, providing indirect evidence of catalytic activity whereby soluble Cu(II) complexes are reduced and precipitated during pretreatment. The concentration of alkali-soluble polymeric and oligomeric lignin was substantially higher for the Cu-catalyzed oxidative pretreatment. This alkali-soluble lignin content increased with time during the catalytic oxidation process, although the molecular weight distributions were unaltered. Yields of aromatic monomers (including phenolic acids and aldehydes) were found to be less than 0.2 % (wt/wt) on lignin. Oxidation of the benzylic alcohol in the lignin side-chain was evident in NMR spectra of the solubilized lignin, whereas minimal changes were observed for the pretreatment-insoluble lignin., Conclusions: These results provide indirect evidence for catalytic activity within the cell wall. The low yields of lignin-derived aromatic monomers, together with the detailed characterization of the pretreatment-soluble and pretreatment-insoluble lignins, indicate that the majority of both lignin pools remained relatively unmodified. As such, the lignins resulting from this process retain features closely resembling native lignins and may, therefore, be amenable to subsequent valorization.

Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.

Nitrous oxide (N2O) is a potent greenhouse gas with a 100-year global warming potential approximately 300 times that of CO2. Because microbes account for over 75% of the N2O released in the U.S., understanding the biochemical processes by which N2O is produced is critical to our efforts to mitigate climate change. In the current study, we used gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to measure the δ(15)N, δ(18)O, δ(15)N(α), and δ(15)N(β) of N2O generated by purified fungal nitric oxide reductase (P450nor) from Histoplasma capsulatum. The isotope values were used to calculate site preference (SP) values (difference in δ(15)N between the central (α) and terminal (β) N atoms in N2O), enrichment factors (ε), and kinetic isotope effects (KIEs). Both oxygen and N(α) displayed normal isotope effects during enzymatic NO reduction with ε values of -25.7‰ (KIE = 1.0264) and -12.6‰ (KIE = 1.0127), respectively. However, bulk nitrogen (average δ(15)N of N(α) and N(β)) and N(β) exhibited inverse isotope effects with ε values of 14.0‰ (KIE = 0.9862) and 36.1‰ (KIE = 0.9651), respectively. The observed inverse isotope effect in δ(15)N(β) is consistent with reversible binding of the first NO in the P450nor reaction mechanism. In contrast to the constant SP observed during NO reduction in microbial cultures, the site preference measured for purified H. capsulatum P450nor was not constant, increasing from ∼ 15‰ to ∼ 29‰ during the course of the reaction. This indicates that SP for microbial cultures can vary depending on the growth conditions, which may complicate source tracing during microbial denitrification.

Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b., (© 2014 Hildenbeutel et al.)

We successfully tested the viability of using synchrotron-based full-field infrared imaging to study biochemical processes inside living cells. As a model system, we studied fibroblast cells exposed to a medium highly enriched with D2O. We could show that the experimental technique allows us to reproduce at the cellular level measurements that are normally performed on purified biological molecules. We can obtain information about lipid conformation and distribution, kinetics of hydrogen/deuterium exchange, and the formation of concentration gradients of H and O isotopes in water that are associated with cell metabolism. The implementation of the full field technique in a sequential imaging format gives a description of cellular biochemistry and biophysics that contains both spatial and temporal information., (Copyright © 2014. Published by Elsevier B.V.)

Background: One route for producing cellulosic biofuels is by the fermentation of lignocellulose-derived sugars generated from a pretreatment that can be effectively coupled with an enzymatic hydrolysis of the plant cell wall. While woody biomass exhibits a number of positive agronomic and logistical attributes, these feedstocks are significantly more recalcitrant to chemical pretreatments than herbaceous feedstocks, requiring higher chemical and energy inputs to achieve high sugar yields from enzymatic hydrolysis. We previously discovered that alkaline hydrogen peroxide (AHP) pretreatment catalyzed by copper(II) 2,2΄-bipyridine complexes significantly improves subsequent enzymatic glucose and xylose release from hybrid poplar heartwood and sapwood relative to uncatalyzed AHP pretreatment at modest reaction conditions (room temperature and atmospheric pressure). In the present work, the reaction conditions for this catalyzed AHP pretreatment were investigated in more detail with the aim of better characterizing the relationship between pretreatment conditions and subsequent enzymatic sugar release., Results: We found that for a wide range of pretreatment conditions, the catalyzed pretreatment resulted in significantly higher glucose and xylose enzymatic hydrolysis yields (as high as 80% for both glucose and xylose) relative to uncatalyzed pretreatment (up to 40% for glucose and 50% for xylose). We identified that the extent of improvement in glucan and xylan yield using this catalyzed pretreatment approach was a function of pretreatment conditions that included H2O2 loading on biomass, catalyst concentration, solids concentration, and pretreatment duration. Based on these results, several important improvements in pretreatment and hydrolysis conditions were identified that may have a positive economic impact for a process employing a catalyzed oxidative pretreatment. These improvements include identifying that: (1) substantially lower H2O2 loadings can be used that may result in up to a 50-65% decrease in H2O2 application (from 100 mg H2O2/g biomass to 35-50 mg/g) with only minor losses in glucose and xylose yield, (2) a 60% decrease in the catalyst concentration from 5.0 mM to 2.0 mM (corresponding to a catalyst loading of 25 μmol/g biomass to 10 μmol/g biomass) can be achieved without a subsequent loss in glucose yield, (3) an order of magnitude improvement in the time required for pretreatment (minutes versus hours or days) can be realized using the catalyzed pretreatment approach, and (4) enzyme dosage can be reduced to less than 30 mg protein/g glucan and potentially further with only minor losses in glucose and xylose yields. In addition, we established that the reaction rate is improved in both catalyzed and uncatalyzed AHP pretreatment by increased solids concentrations., Conclusions: This work explored the relationship between reaction conditions impacting a catalyzed oxidative pretreatment of woody biomass and identified that significant decreases in the H2O2, catalyst, and enzyme loading on the biomass as well as decreases in the pretreatment time could be realized with only minor losses in the subsequent sugar released enzymatically. Together these changes would have positive implications for the economics of a process based on this pretreatment approach.

Copper(II) 2,2'-bipyridine (Cu(II) (bpy))-catalyzed alkaline hydrogen peroxide (AHP) pretreatment was performed on three biomass feedstocks including alkali pre-extracted switchgrass, silver birch, and a hybrid poplar cultivar. This catalytic approach was found to improve the subsequent enzymatic hydrolysis of plant cell wall polysaccharides to monosaccharides for all biomass types at alkaline pH relative to uncatalyzed pretreatment. The hybrid poplar exhibited the most significant improvement in enzymatic hydrolysis with monomeric sugar release and conversions more than doubling from 30% to 61% glucan conversion, while lignin solubilization was increased from 36.6% to 50.2% and hemicellulose solubilization was increased from 14.9% to 32.7%. It was found that Cu(II) (bpy)-catalyzed AHP pretreatment of cellulose resulted in significantly more depolymerization than uncatalyzed AHP pretreatment (78.4% vs. 49.4% decrease in estimated degree of polymerization) and that carboxyl content the cellulose was significantly increased as well (fivefold increase vs. twofold increase). Together, these results indicate that Cu(II) (bpy)-catalyzed AHP pretreatment represents a promising route to biomass deconstruction for bioenergy applications., (Copyright © 2012 Wiley Periodicals, Inc.)

Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (μLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.

H(2) generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H(2) from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O(2) sensitive. Certain filamentous cyanobacteria protect nitrogenase from O(2) by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P(hetN). Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O(2).

Hydrogenases catalyze the reversible formation of H(2), and they are key enzymes in the biological cycling of H(2). H isotopes have the potential to be a very useful tool in quantifying hydrogen ion trafficking in biological H(2) production processes, but there are several obstacles that have thus far limited the application of this tool. Here, we describe a new method that overcomes some of these barriers and is specifically designed to measure isotopic fractionation during enzyme-catalyzed H(2) evolution. A key feature of this technique is that purified hydrogenases are employed, allowing precise control over the reaction conditions and therefore a high level of precision. In addition, a custom-designed high-throughput gas chromatograph/isotope ratio mass spectrometer is employed to measure the isotope ratio of the H(2). Using our new approach, we determined that the fractionation factor for H(2) production by the [NiFe]-hydrogenase from Desulfovibrio fructosovorans is 0.273 ± 0.006. This result indicates that, as expected, protons are highly favored over deuterium ions during H(2) evolution. Potential applications of this newly developed method are discussed., (Copyright © 2011 John Wiley & Sons, Ltd.)

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus., Competing Interests: CB and KKN are on the advisory board of Aurora Alga. AV was supported in part by a grant from Aurora Alga.

[FeFe]-Hydrogenases are complex metalloproteins that catalyze the reversible reduction of protons to molecular hydrogen utilizing a unique diiron subcluster bridged to a [4Fe4S] subcluster. Extensive studies have concentrated on the nature and catalytic activity of the active site, yet relatively little information is available concerning the mechanism of proton transport that is required for this activity. Previously, structural characterization of [FeFe]-hydrogenase from Clostridium pasteurianum indicated a potential proton transport pathway involving four residues (Cys-299, Glu-279, Ser-319, and Glu-282) that connect the active site to the enzyme surface. Here, we demonstrate that substitution of any of these residues resulted in a drastic reduction in hydrogenase activity relative to the native enzyme, supporting the importance of these residues in catalysis. Inhibition studies of native and amino acid-substituted enzymes revealed that Zn(2+) specifically blocked proton transfer by binding to Glu-282, confirming the role of this residue in the identified pathway. In addition, all four of these residues are strictly conserved, suggesting that they may form a proton transport pathway that is common to all [FeFe]-hydrogenases.

Proton transfer to the [Fe-Fe](H) sub-cluster in the Desulfovibrio desulfuricans (DdH) and Clostridium pasteurianum (CpI) [Fe-Fe] hydrogenases was investigated by a combination of first principles and empirical molecular dynamics simulations. Pathways that can be inferred from the X-ray crystal structures of DdH and CpI, i.e., (Glu159→Ser198→Glu156→water460→Cys178→DTMA([Fe-Fe](H)) and (Glu282→Ser319→Glu279→water612→Cys299), respectively, were considered. Proton transfer from Cys178 to DTMA in the [Fe-Fe](H) sub-cluster in DdH was readily observed in our results, specifically when [Fe-Fe](H) was in the reduced state ([Fe(I)-Fe(I)]) or in the mixed valence state for the protonated distal iron Fe(d) ([Fe(I)-Fe(II)-H(-)](H)). A concerted mechanism is proposed, where proton transfer in DdH from Glu159 to Glu156 via Ser198 and Glu156 to Cys178 via water460 readily occurred, as well as from Glu282 to Glu279 via Ser319 and Glu279 to Cys299 via water612 in CpI. The theoretical prediction of the proton transfer characteristics is consistent with the assumed biocatalytic mechanism of the [Fe-Fe] hydrogenases in which the proton binds at Fe(d), providing confirmation that has not been explored so far. The computational results were qualitatively validated by the agreement with experimental hydrogen production activity data for mutated CpI enzymes, relative to the wild-type protein. Finally, the insight provided by the simulations, combined, in part, with experimental validation, are important for establishing an approach in future exploration of proton transfer to the active site in this class of enzymes, and possibly also for biomimetic analogs., (Published by Elsevier B.V.)

Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

The distal nickel site of acetyl-CoA synthase (Ni(d)-ACS) and reduced nickel superoxide dismutase (Ni-SOD) display similar square-planar Ni(II)N(2)S(2) coordination environments. One difference between these two sites, however, is that the nickel ion in Ni-SOD contains a mixed amine/amidate coordination motif while the Ni(d) site in Ni-ACS contains a bisamidate coordination motif. To provide insight into the consequences of the different coordination environments on the properties of the Ni ions, we systematically examined two square-planar Ni(II)N(2)S(2) complexes, one with bisthiolate-bisamidate ligation (Et(4)N)(2)(Ni(L1)).2H(2)O (2) [H(4)L1 = N-(2-mercaptoacetyl)-N'-(2-mercaptoethyl)glycinamide] and another with bisthiolate-amine/amidate ligation K(Ni(HL2)) (3) [H(4)L2 = N-(2''-mercaptoethyl)-2-((2'-mercaptoethyl)amino)acetamide]. Although these two complexes differ only by a single amine versus amidate ligand, their chemical properties are quite different. The stronger in-plane ligand field in the bisamidate complex (Ni(II)(L1))(2-) (2) results in an increase in the energies of the d --> d transitions and a considerably more negative oxidation potential. Furthermore, while the bisamidate complex (Ni(II)(L1))(2-) (2) readily forms a trinuclear species (Et(4)N)(2)({Ni(L1)}(2)Ni).H(2)O (1) and reacts rapidly with O(2), presumably via sulfoxidation, the mixed amine/amidate complex (Ni(II)(HL2))(-) (3) remains monomeric and is stable for days in air. Interestingly, the Ni(III) species of the bisamidate complex formed by chemical oxidation with I(2) can be detected by electron paramagnetic resonance (EPR) spectroscopy while the mixed amine/amidate complex immediately decomposes upon oxidation. To explain these experimentally observed properties, we performed S K-edge X-ray absorption spectroscopy and low-temperature (77 K) electronic absorption measurements as well as both hybrid density functional theory (hybrid-DFT) and spectroscopy oriented configuration interaction (SORCI) calculations. These studies demonstrate that the highest occupied molecular orbital (HOMO) of the bisamidate complex (Ni(II)(L1))(2-) (2) has more Ni character and is significantly destabilized relative to the mixed amine/amidate complex (Ni(II)(HL2))(-) (3) by approximately 6.2 kcal mol(-1). The consequence of this destabilization is manifested in the nucleophilic activation of the doubly filled HOMO, which makes (Ni(II)(L1))(2-) (2) significantly more reactive toward electrophiles such as O(2).

The assembly and activity of cytochrome c oxidase is dependent on the availability of heme A, one of its essential cofactors. In eukaryotes, two inner mitochondrial membrane proteins, heme O synthase (Cox10) and heme A synthase (Cox15), are required for heme A biosynthesis. In this report, we demonstrate that in Saccharomyces cerevisiae the transcription of COX15 is regulated by Hap1, a transcription factor whose activity is positively controlled by intracellular heme concentration. Conversely, COX10, the physiological partner of COX15, does not share the same regulatory mechanism with COX15. Interestingly, protein quantification identified an 8:1 protein ratio between Cox15 and Cox10. Together, these results suggest that heme A synthase and/or heme O synthase might play a new, unidentified role in addition to heme A biosynthesis.

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803-806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.

Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [Delta7-H3m](2), is a four-alpha-helix bundle that contains two bis(3-methyl-l-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-l-histidine) heme binding sites in [Delta7-H3m](2). Spectroscopic analyses indicate that 2 equiv of each heme binds to [Delta7-H3m](2), as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.

The transport of water into and out of unicellular organisms is a seemingly simple process in which water diffuses either through pores or directly across the membrane. Because of the rates at which water can theoretically diffuse, it is generally accepted that intracellular water is indistinguishable from extracellular water. Using stable isotope ratio mass spectrometry, we directly tested this assumption. Here we demonstrate that under active growth, up to 70% of the intracellular water in log-phase Escherichia coli cells was actually generated during metabolism and was isotopically distinct from extracellular water. The contribution of metabolism to intracellular water was substantially less in stationary-phase or quiescent cells.

Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidase (CcO) enzymes. Despite its obvious importance to CcO and the electron transport pathway, essentially nothing is known concerning the regulation of heme A. Because CcO is the only natural target for heme A and copper is also required for CcO activity, it was postulated that copper might regulate heme A homeostasis. Work reported previously demonstrated that there is often a strong connection between copper and iron homeostasis in general, and circumstantial evidence pointed to a possible specific link between copper and heme A. To address this question, we conducted experiments to determine rigorously whether copper plays a role in heme A homeostasis. The two enzymes responsible for the conversion of heme B to heme A, heme O synthase (HOS) and heme A synthase (HAS), were separately genomically epitope-tagged in Saccharomyces cerevisiae, and their expression under various copper conditions was quantified by Western blot analysis. These results demonstrated that the sum of transcription, translation, and stability of HOS and HAS were independent of copper. Additionally, the effects of intracellular copper concentrations on the activity of HOS and HAS from Bacillus subtilis (expressed in Escherichia coli) and Rhodobacter sphaeroides were examined by analysis of cellular heme extracts. No trends with respect to intracellular copper were observed. In combination, our results demonstrate that intracellular copper levels do not affect the transcription, translation, stability, or activity of either HOS or HAS.

Cytochrome c oxidase requires multiple heme and copper cofactors to catalyze the reduction of molecular oxygen to water. Although significant progress has been made in understanding the transport and incorporation of the copper ions, considerably less is known about the trafficking and insertion of the heme cofactors. Heme O synthase (HOS) and heme A synthase (HAS) from Rhodobacter sphaeroides (Cox10 and Cox15, respectively) and Bacillus subtilis (CtaB and CtaA, respectively) have been cloned and expressed in Escherichia coli. Our results demonstrate that HOS copurifies with HAS and that HAS copurifies with HOS, indicating that HOS and HAS interact and may form a physiologically relevant complex in vivo. Consistent with this hypothesis, the presence of HAS alters the total level of farnesylated hemes, providing further evidence that HOS and HAS interact. Our current working model is that HOS and HAS form a complex and that heme O is transferred directly from HOS to HAS. Because of the strong sequence similarity and evolutionary relationship between R. sphaeroides and mitochondria, our data suggest that this complex may form in eukaryotes as well.

The bifunctional enzyme carbon monoxide dehydrogenase/acetyl-coenzyme A (CoA) synthase (CODH/ACS) is a key enzyme in the Wood-Ljungdahl pathway of carbon fixation. Carbon monoxide is combined with a methyl group and ultimately converted to acetyl-CoA at a unique Ni-containing bimetallic site in the A-cluster of this enzyme. Despite years of extensive biochemical and spectroscopic studies and the recent report of three separate crystal structures, the mechanism by which acetyl-CoA is synthesized is still unknown. Over the past two years there have been a number of significant developments regarding ACS. This Account critically examines these recent developments and especially focuses on those areas that are still a matter of debate.

Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidases. The final step in heme A biosynthesis requires the oxidation of the C8 methyl substituent on pyrrole ring D to an aldehyde, a reaction catalyzed by heme A synthase. To effect this transformation, heme A synthase is proposed to utilize a heme B cofactor, oxidizing the substrate via successive monooxygenase reactions. Consistent with this hypothesis, the activity of heme A synthase is found to be strictly dependent on molecular oxygen. Surprisingly, when cells expressing heme A synthase were incubated with (18)O(2), no significant incorporation of label was observed in heme A, the C8 alcohol intermediate, or the C8 overoxidized byproduct. Conversely, when the cells were grown in H(2)(18)O, partial labeling was observed at every heme oxygen position. These results suggest that the oxygen on the heme A aldehyde is derived from water. Although our data do not allow us to exclude the possibility of exchange with water inside of the cell, the results seem to question a mechanism utilizing successive monooxygenase reactions and support instead a mechanism of heme O oxidation via electron transfer.

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) utilizes a unique Ni-M bimetallic site in the biosynthesis of acetyl-CoA, where a square-planar Ni ion is coordinated to two thiolates and two deprotonated amides in a Cys-Gly-Cys motif. The identity of M is currently a matter of debate, although both Cu and Ni have been proposed. In an effort to model ACS's unusual active site and to provide insight into the mechanism of acetyl-CoA formation and the role of each of the metals ions, we have prepared and structurally characterized a number of Ni(II)-peptide mimic complexes. The mononuclear complexes Ni(II) N, N'-bis(2-mercaptoethyl)oxamide (1), Ni(II) N, N'-ethylenebis(2-mercaptoacetamide) (2), and Ni(II) N, N'-ethylenebis(2-mercaptopropionamide) (3) model the Ni(Cys-Gly-Cys) site and can be used as synthons for additional multinuclear complexes. Reaction of 2 with MeI resulted in the alkylation of the sulfur atoms and the formation of Ni(II) N, N'-ethylenebis(2-methylmercaptoacetamide) (4), demonstrating the nucleophilicity of the terminal alkyl thiolates. Addition of Ni(OAc)(2).4H(2)O to3 resulted in the formation of a trinuclear species (5), while 2 crystallizes as an unusual paddlewheel complex (6) in the presence of nickel acetate. The difference in reactivity between the similar complexes 2 and 3 highlights the importance of ligand design when synthesizing models of ACS. Significantly,5 maintains the key features observed in the active site of ACS, namely a square-planar Ni coordinated to two deprotonated amides and two thiolates, where the thiolates bridge to a second metal, suggesting that 5 is a reasonable structural model for this unique enzyme.