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Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).

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Flexible carbon fiber cloth (CFC) is an important scaffold and/or current collector for active materials in the development of flexible self-supportive electrode materials (SSEMs), especially in lithium-ion batteries. However, during the intercalation of Li ions into the matrix of CFC (below 0.5 V vs. Li/ Li + ), the incompatibility in the capacity of the CFC, when used directly as an

Interfacial engineering and elemental doping are the two parameters to enhance the catalytic behavior of cobalt nitrides for the alkaline hydrogen evolution reaction (HER). However, simultaneously combining these two parameters to improve the HER catalytic properties of cobalt nitrides in alkaline media is rarely reported and also remains challenging in acidic media. Herein, it is demonstrated that high‐valence non‐3d metal and non‐metal integration can simultaneously achieve Co‐based nitride/oxide interstitial compound phase boundaries on stainless steel mesh (denoted Mo‐Co5.47N/N‐CoO) for efficient HER in alkaline and acidic media. Density functional theory (DFT) calculations show that the unique structure does not only realize multi‐active sites, enhanced water dissociation kinetics, and low hydrogen adsorption free energy in alkaline media, but also enhances the positive charge density of hydrogen ions (H+) to effectively allow H+ to receive electrons from the catalysts surface toward promoting the HER in acidic media. As a result, the as‐prepared Mo‐Co5.47N/N‐CoO demands HER overpotential of −28 mV@10 mA cm−2 in an alkaline medium, and superior to the commercial Pt/C at a current density > 44 mA cm−2 in acidic medium. This work paves a useful strategy to design efficient cobalt‐based electrocatalysts for HER and beyond.

In vitro, protein disulfide isomerase (Pdi1p) introduces disulfides into proteins (oxidase activity) and provides quality control by catalyzing the rearrangement of incorrect disulfides (isomerase activity). Protein disulfide isomerase (PDI) is an essential protein in Saccharomyces cerevisiae, but the contributions of the catalytic activities of PDI to oxidative protein folding in the endoplasmic reticulum (ER) are unclear. Using variants of Pdi1p with impaired oxidase or isomerase activity, we show that isomerase-deficient mutants of PDI support wild-type growth even in a strain in which all of the PDI homologues of the yeast ER have been deleted. Although the oxidase activity of PDI is sufficient for wild-type growth, pulse-chase experiments monitoring the maturation of carboxypeptidase Y reveal that oxidative folding is greatly compromised in mutants that are defective in isomerase activity. Pdi1p and one or more of its ER homologues (Mpd1p, Mpd2p, Eug1p, Eps1p) are required for efficient carboxypeptidase Y maturation. Consistent with its function as a disulfide isomerase in vivo, the active sites of Pdi1p are partially reduced (32 ؎ 8%) in vivo. These results suggest that PDI and its ER homologues contribute both oxidase and isomerase activities to the yeast ER. The isomerase activity of PDI can be compromised without affecting growth and viability, implying that yeast proteins that are essential under laboratory conditions may not require efficient disulfide isomerization.Disulfide bonds provide added stability to extracellular proteins by covalently cross-linking two cysteines. Disulfide formation is often error-prone, particularly in the early stages of folding (1), and pairing the correct cysteines into disulfides requires that any mispaired disulfides must be broken and reformed in a different configuration to reach the native structure. In bacteria, disulfides are formed in the periplasm by an elaborate system of oxidases and isomerases that assure the correct cysteines are connected (2, 3). In eukaryotes this posttranslational modification occurs in the endoplasmic reticulum (ER) 1 where a complex set of enzyme catalysts promotes correct disulfide formation. In yeast (4, 5) and mammalian cells (6) the oxidizing equivalents for disulfide formation are generated principally by Ero1p. These disulfides in turn are delivered to protein disulfide isomerase (Pdi1p), an essential folding catalyst of the endoplasmic reticulum (7).Both yeast and mammalian protein disulfide isomerase (PDI) are composed of four domains (termed a, b, b, and a) and an anionic tail (c) (8). The two catalytic domains (a and a) are located at the ends of the molecule, and each contains an active site with the sequence CGHC. The catalytic thioredoxin domains are separated by two non-catalytic thioredoxin domains (b and b) (9) in a multidomain structure (abbac). When the active-site cysteines of PDI are in a disulfide (oxidized) form, the enzyme can introduce disulfides into proteins (oxidase activity) through thiol/disulfide exchange...

Protein-disulfide isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum. PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange. In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines whose location and separation distance varies by organism. In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids. In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence. By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable even in a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold. Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro.Cell surface and secreted proteins are required to function in a more oxidizing environment than that of the cytoplasm and, as a result, often contain disulfide bonds that covalently link two cysteines and impart structural stability in the harsh environment of the cell exterior. In vitro, the formation of these disulfides is often slow and error-prone (1). In eukaryotes, disulfide bond formation and rearrangement occurs in the endoplasmic reticulum (ER), 1 where it is catalyzed by proteindisulfide isomerase (PDI) (1-3), an essential, abundant, and ubiquitously expressed 55-kDa resident ER protein. Structurally, PDI is composed of four thioredoxin motifs. The rat protein exhibits a quite elongated shape in solution (axial ratio ϭ 5.7), suggesting that the domains are extended and arranged linearly, at least in the ultracentrifuge (4). The thioredoxin domains near the N and C termini termed the a and aЈ domains, respectively, contain active sites, each with a sequence CXXC that facilitates thiol-disulfide exchange. The catalytic domains are separated by two non-catalytic domains, b and bЈ, and the multi-domain structure terminates in a highly acidic C-terminal tail termed the c domain (abbЈaЈc). The bЈ domain and the interface between the bЈ and aЈ domains contribute to peptide and protein substrate interactions (5).In addition to its four active site cysteines, PDI contains two internal, non-active site cysteines whose function has been unclear. In mammals and birds, the internal cysteines are conserved, located in the bЈ domain of the protein, and are separated by 30 amino acids. In yeast and fungi including Schizosaccharomyces pombe, the non-active site cysteines are located near the N-terminal active site in the a domain ...

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