Isolation and characterization of a new [FeFe]-hydrogenase from Clostridium perfringens

© 2015 International Union of Biochemistry and Molecular Biology, Inc. This paper reports the first characterization of an [FeFe]-hydrogenase from a Clostridium perfringens strain previously isolated in our laboratory from a pilot-scale bio-hydrogen plant that efficiently produces H2 from waste biomasses. On the basis of sequence analysis, the enzyme is a monomer formed by four domains hosting various iron–sulfur centres involved in electron transfer and the catalytic center H-cluster. After recombinant expression in Escherichia coli, the purified protein catalyzes H2 evolution at high rate of 1645±16s−1. The optimal conditions for catalysis are in the pH range 6.5–8.0 and at the temperature of 50°C. EPR spectroscopy showed that the H-cluster of the oxidized enzyme displays a spectrum coherent with the Hox state, whereas the CO-inhibited enzyme has a spectrum coherent with the Hox-CO state. FTIR spectroscopy showed that the purified enzyme is composed of a mixture of redox states, with a prevalence of the Hox; upon reduction with H2, vibrational modes assigned to the Hred state were more abundant, whereas binding of exogenous CO resulted in a spectrum assigned to the Hox-CO state. The spectroscopic features observed are similar to those of the [FeFe]-hydrogenases class, but relevant differences were observed given the different protein environment hosting the H-cluster

Построение модели компетентности экспертов

Представлено програму реалізації моделі компетентності експертів за наслідками кластерного аналізу.The aim is to develop research and methods of processing expertise and the method of selection of a competent team of experts using simulation. Expert methods are based solely on expert assessments made on the problem to study. This mechanism producing these estimates remain uncertain. Typically, it is unknown even to the expert, is entirely individual, personal character and can not be repeated or replicated by someone else. The basis using expert techniques are deep specialist knowledge and skills to summarize your experience and global research and development on a particular issue. The problem processing the results of the examination group - a set of tasks, each of which solution to some extent depends on the purpose of the examination conducted. However, there are some "eternal" issues that need to be solve in processing expert information. One of these issues - competency assessment expert. Scientific novelty of the proposed approach to determining quantitative assessments of competence of the expert and their expert use for processing data. Then, a simulation model was built with three divisions: Gauss, Laplace and linear. The work proved theoretically should look like logistic curve, which depends on the distance to the center of the cluster.Представлена программа реализации модели компетентности экспертов согласно результатами кластерного анализа

Proton-Coupled Reduction of the Catalytic [4Fe-4S] Cluster in [FeFe]-Hydrogenases

In nature, [FeFe]-hydrogenases catalyze the uptake and release of molecular hydrogen (H2) at a unique iron-sulfur cofactor. The absence of an electrochemical overpotential in the H2 release reaction makes [FeFe]-hydrogenases a prime example of efficient biocatalysis. However, the molecular details of hydrogen turnover are not yet fully understood. Herein, we characterize the initial one-electron reduction of [FeFe]-hydrogenases by infrared spectroscopy and electrochemistry and present evidence for proton- coupled electron transport during the formation of the reduced state Hred′. Charge compensation stabilizes the excess electron at the [4Fe-4S] cluster and maintains a conservative configuration of the diiron site. The role of Hred′ in hydrogen turnover and possible implications on the catalytic mechanism are discussed. We propose that regulation of the electronic properties in the periphery of metal cofactors is key to orchestrating multielectron processes

A pulsed EPR study of clustering of Yb3+ ions incorporated in GeO2 glass

The structural aspects of clustering of Yb3+ ions and the paramagnetic behavior of these clusters have been investigated in GeO 2 glasses doped with 140-1100 ppm by weight of Yb2O 3 using time-domain electron paramagnetic resonance (EPR) spectroscopic techniques. The echo-detected EPR (EDEPR) spectra of Yb 3+ ions and their unusual dependencies on microwave power and magnetic field have been found to be indicative of the formation of clusters of these rare earth ions in GeO2 glass that behave as non-Kramers type spin systems. The magnetic field and concentration dependence of phase relaxation rates of Yb3+ in these glasses further substantiate such a scenario and indicate the formation of clusters of Yb3+ ions. A comparison of the EDEPR spectra with calculated cw-EPR line shapes yields a semi-quantitative measure of the typical cluster size of ≥3 Yb atoms and intra-cluster Yb-Yb distances of 3.5-4.0 Å in these glasses at doping levels of ≥350 ppm of Yb2O3. © 2003 Elsevier B.V. All rights reserved

A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster

Bridging-hydride influence on the electronic structure of an [FeFe] hydrogenase active-site model complex revealed by XAES-DFT

[[abstract]]Two crystallized [FeFe] hydrogenase model complexes, 1 = (μ-pdt)[Fe(CO)2(PMe3)]2 (pdt = SC1H2C2H2C3H2S), and their bridging-hydride (Hy) derivative, [1Hy]+++ = [(μ-H)(μ-pdt)[Fe(CO)2 (PMe3)]2]+ (BF4−), were studied by Fe K-edge X-ray absorption and emission spectroscopy, supported by density functional theory. Structural changes in [1Hy]+++ compared to 1 involved small bond elongations (<0.03 Å) and more octahedral Fe geometries; the Fe–H bond at Fe1 (closer to pdt-C2) was [similar]0.03 Å longer than that at Fe2. Analyses of (1) pre-edge absorption spectra (core-to-valence transitions), (2) Kβ1,3, Kβ′, and Kβ2,5 emission spectra (valence-to-core transitions), and (3) resonant inelastic X-ray scattering data (valence-to-valence transitions) for resonant and non-resonant excitation and respective spectral simulations indicated the following: (1) the mean Fe oxidation state was similar in both complexes, due to electron density transfer from the ligands to Hy in [1Hy]+++. Fe 1s→3d transitions remained at similar energies whereas delocalization of carbonyl AOs onto Fe and significant Hy-contributions to MOs caused an [similar]0.7 eV up-shift of Fe1s→(CO)s,p transitions in [1Hy]+++. Fed-levels were delocalized over Fe1 and Fe2 and degeneracies biased to Oh–Fe1 and C4v–Fe2 states for 1, but to Oh–Fe1,2 states for [1Hy]+++. (2) Electron-pairing of formal Fe(d7) ions in low-spin states in both complexes and a higher effective spin count for [1Hy]+++ were suggested by comparison with iron reference compounds. Electronic decays from Fe d and ligand s,p MOs and spectral contributions from Hys,p→1s transitions even revealed limited site-selectivity for detection of Fe1 or Fe2 in [1Hy]+++. The HOMO/LUMO energy gap for 1 was estimated as 3.0 ± 0.5 eV. (3) For [1Hy]+++ compared to 1, increased Fed (x2 − y2) − (z2) energy differences ([similar]0.5 eV to [similar]0.9 eV) and Fed→d transition energies ([similar]2.9 eV to [similar]3.7 eV) were assigned. These results reveal the specific impact of Hy-binding on the electronic structure of diiron compounds and provide guidelines for a directed search of hydride species in hydrogenases.[[notice]]補正完畢[[journaltype]]國外[[ispeerreviewed]]Y[[booktype]]紙本[[booktype]]電子版[[countrycodes]]GB

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. Principal Findings: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.United States. Dept. of Energy. (contract DE-AC36-08-GO28308

Investigation of the Stationary and Transient A1·− Radical in Trp → Phe Mutants of Photosystem I

Photosystem I (PS I) contains two symmetric branches of electron transfer cofactors. In both the A- and B-branches, the phylloquinone in the A1 site is π-stacked with a tryptophan residue and is H-bonded to the backbone nitrogen of a leucine residue. In this work, we use optical and electron paramagnetic resonance (EPR) spectroscopies to investigate cyanobacterial PS I complexes, where these tryptophan residues are changed to phenylalanine. The time-resolved optical data show that backward electron transfer from the terminal electron acceptors to P700·+ is affected in the A- and B-branch mutants, both at ambient and cryogenic temperatures. These results suggest that the quinones in both branches take part in electron transport at all temperatures. The electron-nuclear double resonance (ENDOR) spectra of the spin-correlated radical pair P700·+A1·− and the photoaccumulated radical anion A1·−, recorded at cryogenic temperature, allowed the identification of characteristic resonances belonging to protons of the methyl group, some of the ring protons and the proton hydrogen-bonded to phylloquinone in the wild type and both mutants. Significant changes in PS I isolated from the A-branch mutant are detected, while PS I isolated from the B-branch mutant shows the spectral characteristics of wild-type PS I. A possible short-lived B-branch radical pair cannot be detected by EPR due to the available time resolution; therefore, only the A-branch quinone is observed under conditions typically employed for EPR and ENDOR spectroscopies