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[NiFeSe] hydrogenases are a subgroup of the large family of [NiFe] hydrogenases in which a selenocysteine ligand coordinates the Ni atom at the active site. As observed for other selenoproteins, the [NiFeSe] hydrogenases display much higher catalytic activities than their Cys-containing homologues. Here, we review the biochemical, catalytic, spectroscopic and structural properties of known [NiFeSe] hydrogenases, namely from the Hys (group 1 [NiFeSe] hydrogenase), Fru (F420-reducing [NiFeSe] hydrogenases) and Vhu families (F420-non-reducing [NiFeSe] hydrogenases). A survey of new [NiFeSe] hydrogenases present in the databases showed that all enzymes belong to either group 1 periplasmic uptake hydrogenases (Hys) or to group 3 cytoplasmic hydrogenases (Fru and Vhu) and are present in either sulfate-re-ducing or methanogenic microorganisms. In both kinds of organisms, the [NiFeSe] hydrogenases are preferred over their Cys-containing homologues if selenium is available. Since no structural information is available for the Vhu and Fru enzymes, we have modelled the large subunit of these enzymes and analyzed the area surrounding the active site. Three [NiFeSe] hydrogenases of the Hys and Vhu types were identified in which the selenocysteine residue is found in a different location in the sequence, which could result in a different coordination to the Ni atom. The high activity and fast reactivation, together with a degree of oxygen tolerance for the H2-production activity, make the Hys hydrogenases attractive catalysts for technological applications. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA. ; This work was supported by research grants PTDC/BIAPRO/70429/2006 funded by Fundação para a Ciência e Tecnologia (FCT, MCES, Portugal) and European Union FEDER program, and CTQ2006-12097 funded by the Ministerio de Ciencia e Innovacion (Spain). ; Peer Reviewed

25 páginas, 9 figuras y 2 tablas ; The binding of carbon monoxide a competitive inhibitor of many hydrogenases, to the active site of Desulfovibrio fructosovorans hydrogenase has been studied by infrared spectroscopy in a spectroelectrochemical cell. Direct evidence has been obtained of which redox states of the enzyme can bind extrinsic CO. Redox states A, B and SU do not bind extrinsic CO. only after reductive activation of the hydrogenase can CO bind to the active site. Two states with bound extrinsic CO can be distinguished by FTIR. These two states are in redox equilibrium and are most probably due to different oxidation states of the proximal 4Fe-4S cluster. Vibrational frequencies and theoretical quantum mechanics studies (DFT) of this process preclude the possibility of strong bonding of extrinsic CO to the Fe or Ni atoms or the site. We propose that CO inhibition is caused by weak interaction of the extrinsic ligand with the Ni atom, blocking electron and proton transfer at the active site. A calculated structure with a weakly bound extrinsic CO at Ni has relative CO frequencies in excellent agreement with the experimental ones. ; The Spanish Ministry of Science and Technology (project BQU2000-0991) and the European Union BIOTECH program (grant BIO-98-0280) supported this work. We thank Prof. Siem Albracht for useful discussions, Dr. Arturo Martinez-Arias for doing the EPR measurements, the Autonomous Community of Madrid (CAM) for the postdoctoral fellowship of A. L. De Lacey and the German Academic Exchange Service (DAAD) for the postdoctoral fellowship of C. Stadler. MBH thanks NSF (CHE-9800184) for financial support. ; Peer reviewed

The successive steps of laccase immobilization on chemically modified Au electrodes were monitored using ATR-SEIRAS and in situ STM. Successful covalent immobilization of the enzyme on Au electrodes modified by a mixed aminophenyl-mercaptohexanol adlayer and on Au electrodes modified by a 4-aminothiophenyl SAM via a Schiff base reaction followed by the formation of amide bonds is revealed by the emergence of the corresponding bands in the ATR-SEIRA spectra, and an enzyme coverage on aminophenyl-mercaptohexanol- modified Au electrodes of about (7.27 ± 1.93) × 10 11 laccase units per cm 2 was calculated from STM images. The small differences between the ATR-SEIRA spectra of the enzyme immobilized on aminophenyl-mercaptohexanol-modified Au electrodes and the ATR-SEIRA spectra of the enzyme immobilized on 4-aminothiophenyl-modified Au electrodes are attributed to a different orientation of the immobilized enzyme due to the presence on the surface of aminophenyl-mercaptohexanol-modified Au electrodes of OH functional groups that favor an orientation of laccase with the Cu T1 center of the enzyme facing the electrode surface, thus, allowing a high activity for direct electrocatalysis of the ORR at low overpotentials. © 2012 American Chemical Society. ; This work has been funded by the DGI (Ministerio de Ciencia e Innovación), under Projects CTQ2009-07017 and CTQ2009-12649, and by the European Union (FP7 Project "3D-Nanobiodevice", NMP4-SL-2009-229255). C.V.-D. acknowledges a JAE-Doc fellowship from CSIC, and M.P. acknowledges the Ramon y Cajal 2009 program from the Spanish MICINN. ; Peer Reviewed

High surface area graphene electrodes were prepared by simultaneous electrodeposition and electroreduction of graphene oxide. The electrodeposition process was optimized in terms of pH and conductivity of the solution and the obtained graphene electrodes were characterized by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy and electrochemical methods (cyclic voltammetry and impedance spectroscopy). Electrodeposited electrodes were further functionalized to carry out covalent immobilization of two oxygen-reducing multicopper oxidases: laccase and bilirubin oxidase. The enzymatic electrodes were tested as direct electron transfer based biocathodes and catalytic currents as high as 1 mA/cm2 were obtained. Finally, the mechanism of the enzymatic oxygen reduction reaction was studied for both enzymes calculating the Tafel slopes and transfer coefficients. ; This work has received funding from the European Union's Seventh Framework Programme under grant agreement BIOENERGY, FP7-PEOPLE-2013-ITN-607793. ; Peer Reviewed

High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification. ; This study was based upon work funded by EU Projects (NMP4-SL-2009-229255-3D-Nanobiodevice, FP7-KBBE-2010-4-26537-Peroxicats and COST Action CM0701) and a project from the Spanish Government (BIO2010-19697). D.M.M. was supported by a JAE grant and D.G.P. was supported by a Peroxicats contract. M.P. received support from the 2009 Ramón y Cajal programme of the Spanish MINECO. ; Peer reviewed