Characterization of the active site and calcium binding in cytochromecnitrite reductases

The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca2+ binding that may have an impact on substrate reduction

Quantum dot interactions with and toxicity to Shewanella oneidensis MR-1

Combining abiotic photosensitisers such as quantum dots (QDs) with non-photosynthetic bacteria presents an intriguing concept into the design of artificial photosynthetic organisms and solar-driven fuel production. Shewanella oneidensis MR-1 (MR-1) is a versatile bacterium concerning respiration, metabolism and biocatalysis, and is a promising organism for artificial photosynthesis as the bacterium's synthetic and catalytic ability provides a potential system for bacterial biohydrogen production. MR-1's hydrogenases are present in the periplasmatic space. It follows that for photoenergised electrons to reach these enzymes, QDs will need to be able to enter the periplasm, or electrons need to enter the periplasm via the Mtr pathway that is responsible for MR-1's extracellular electron transfer ability. As a step towards this goal, various QDs were tested for their photo-reducing potential, nanotoxicology and further for their interaction with MR-1. CdTe/CdS/TGA, CdTe/CdS/Cysteamine, a commercial, negatively charged CdTe and CuInS2/ZnS/PMAL QDs were examined. The photoreduction potential of the QDs was confirmed by measuring their ability to photoreduce methyl viologen with different sacrificial electron donors. The commercial CdTe and CuInS2/ZnS/PMAL QDs showed no toxicity towards MR-1 as evaluated by a colony-forming units method and a fluorescence viability assay. Only the commercial negatively charged CdTe QDs showed good interaction with MR-1. With transmission electron microscopy, QDs were observed both in the cytoplasm and periplasm. These results inform on the possibilities and bottlenecks when developing bionanotechnological systems for the photosynthetic production of biohydrogen by MR-1

Carbon Dots as Versatile Photosensitizers for Solar-Driven Catalysis with Redox Enzymes

Light-driven enzymatic catalysis is enabled by the productive coupling of a protein to a photosensitizer. Photosensitizers used in such hybrid systems are typically costly, toxic, and/or fragile, with limited chemical versatility. Carbon dots (CDs) are low-cost, nanosized light-harvesters that are attractive photosensitizers for biological systems as they are water-soluble, photostable, nontoxic, and their surface chemistry can be easily modified. We demonstrate here that CDs act as excellent light-absorbers in two semibiological photosynthetic systems utilizing either a fumarate reductase (FccA) for the solar-driven hydrogenation of fumarate to succinate or a hydrogenase (H$_{2}$ase) for reduction of protons to H$_{2}$. The tunable surface chemistry of the CDs was exploited to synthesize positively charged ammonium-terminated CDs (CD-NHMe$_{2}$$^{+}$), which were capable of transferring photoexcited electrons directly to the negatively charged enzymes with high efficiency and stability. Enzyme-based turnover numbers of 6000 mol succinate (mol FccA)$^{-1}$ and 43,000 mol H$_{2}$ (mol H$_{2}$ase)$^{-1}$ were reached after 24 h. Negatively charged carboxylate-terminated CDs (CD-CO$_{2}$$^{-}$) displayed little or no activity, and the electrostatic interactions at the CD–enzyme interface were determined to be essential to the high photocatalytic activity observed with CD-NHMe$_{2}$$^{+}$. The modular surface chemistry of CDs together with their photostability and aqueous solubility make CDs versatile photosensitizers for redox enzymes with great scope for their utilization in photobiocatalysis.This work was supported by a Cambridge Australia Poynton PhD scholarship (to G.A.M.H.), the BBSRC (BB/K010220/1 to E.R. and BB/K009885/1 to J.N.B.), an Oppenheimer PhD scholarship (to B.C.M.M.), and a Marie Curie postdoctoral fellowship (GAN 624997 to C.A.C.)

Multilayered lipid membrane stacks for biocatalysis using membrane enzymes

Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules and greatly increase surface area and hence membrane protein concentration. Here we report on a supramolecular assembly of a multilayered lipid membrane system in which poly-L-lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo3 from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane-bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. We deduce by atomic force microscopy, CV and fluorescence microscopy that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and have significant potential for mimicry in biotechnology applications such as energy production or biosensing

High Performance Reduction of H2O2 with an Electron Transport Decaheme Cytochrome on a Porous ITO Electrode

The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm−2 at 0.4 Vvs SHE could be obtained at pH 6.5 (Eonset = 0.72 V). UV−visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors.BBSRC, ERC, DFG, Marie Sklodowska Curie fellowshi

Coherent Electron Transport across a 3 nm Bioelectronic Junction Made of Multi-Heme Proteins

Multi-heme cytochromes (MHCs) are fascinating proteins used by bacterial organisms to shuttle electrons within, between, and out of their cells. When placed in solid-state electronic junctions, MHCs support temperature-independent currents over several nanometers that are 3 orders of magnitude higher compared to other redox proteins of similar size. To gain molecular-level insight into their astonishingly high conductivities, we combine experimental photoemission spectroscopy with DFT+Σ current-voltage calculations on a representative Gold-MHC-Gold junction. We find that conduction across the dry, 3 nm long protein occurs via off-resonant coherent tunneling, mediated by a large number of protein valence-band orbitals that are strongly delocalized over heme and protein residues. This picture is profoundly different from the electron hopping mechanism induced electrochemically or photochemically under aqueous conditions. Our results imply that the current output in solid-state junctions can be even further increased in resonance, for example, by applying a gate voltage, thus allowing a quantum jump for next-generation bionanoelectronic devices

Light-Driven H2 Evolution and C═C or C═O Bond Hydrogenation by Shewanella oneidensis : A Versatile Strategy for Photocatalysis by Nonphotosynthetic Microorganisms

Photocatalytic chemical synthesis by coupling abiotic photosensitizers to purified enzymes provides an effective way to overcome the low conversion efficiencies of natural photosynthesis while exploiting the high catalytic rates and selectivity of enzymes as renewable, earth-abundant electrocatalysts. However, the selective synthesis of multiple products requires more versatile approaches and should avoid the time-consuming and costly processes of enzyme purification. Here we demonstrate a cell-based strategy supporting light-driven H2 evolution or the hydrogenation of C═C and C═O bonds in a nonphotosynthetic microorganism. Methylviologen shuttles photoenergized electrons from water-soluble photosensitizers to enzymes that catalyze H2 evolution and the reduction of fumarate, pyruvate, and CO2 in Shewanella oneidensis. The predominant reaction is selected by the experimental conditions, and the results allow rational development of cell-based strategies to harness nature’s intrinsic catalytic diversity for selective light-driven synthesis of a wide range of products

Serology based disease status of Pakistani population infected with Hepatitis B virus

<p>Abstract</p> <p>Background</p> <p>The infection rate of hepatitis B virus is continuously increasing in Pakistan. Therefore, a comprehensive study of epidemiological data is the need of time.</p> <p>Methods</p> <p>A total of 1300 individuals were screened for HBV infection markers including HBsAg, anti-HBsAg, HBeAg and anti-HBcAg. The association of these disease indicators was compared with patients' epidemiological characteristics like age, socio-economic status and residential area to analyze and find out the possible correlation among these variables and the patients disease status.</p> <p>Results</p> <p>52 (4%) individuals were found positive for HBsAg with mean age 23.5 ± 3.7 years. 9.30%, 33.47% and 12% individuals had HBeAg, antibodies for HBsAg, and antibodies for HBcAg respectively. HBsAg seropositivity rate was significantly associated (<it>p </it>= 0.03) with the residing locality indicating high infection in rural areas. Antibodies titer against HBsAg decreased with the increasing age reflecting an inverse correlation.</p> <p>Conclusion</p> <p>Our results indicate high prevalence rate of Hepatitis B virus infection and nationwide vaccination campaigns along with public awareness and educational programs are needed to be practiced urgently.</p

Functional Organization of Locomotor Interneurons in the Ventral Lumbar Spinal Cord of the Newborn Rat

Although the mammalian locomotor CPG has been localized to the lumbar spinal cord, the functional-anatomical organization of flexor and extensor interneurons has not been characterized. Here, we tested the hypothesis that flexor and extensor interneuronal networks for walking are physically segregated in the lumbar spinal cord. For this purpose, we performed optical recordings and lesion experiments from a horizontally sectioned lumbar spinal cord isolated from neonate rats. This ventral hemi spinal cord preparation produces well-organized fictive locomotion when superfused with 5-HT/NMDA. The dorsal surface of the preparation was visualized using the Ca2+ indicator fluo-4 AM, while simultaneously monitoring motor output at ventral roots L2 and L5. Using calcium imaging, we provided a general mapping view of the interneurons that maintained a stable phase relationship with motor output. We showed that the dorsal surface of L1 segment contains a higher density of locomotor rhythmic cells than the other segments. Moreover, L1 segment lesioning induced the most important changes in the locomotor activity in comparison with lesions at the T13 or L2 segments. However, no lesions led to selective disruption of either flexor or extensor output. In addition, this study found no evidence of functional parcellation of locomotor interneurons into flexor and extensor pools at the dorsal-ventral midline of the lumbar spinal cord of the rat