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Title: Infrared spectroelectrochemical study of E. coli NiFe hydrogenase 1
Author: Hidalgo González, Ricardo
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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This thesis investigates the catalytic mechanism of NiFe hydrogenases. The specific enzyme studied in this work, E. coli Hyd-1, is an efficient catalyst for H2 oxidation even in the presence of O2. A method for studying the chemistry of the active site of this enzyme, under catalytic conditions, is developed. The combination of IR spectroscopy with protein film electrochemistry in situ is demonstrated. This was achieved by adsorbing the hydrogenase on a high surface area carbon nanoparticle electrode; and by the design of a spectroelectrochemical flow cell that provides efficient mass transport conditions. A complete redox characterisation of the active site for a hydrogenase immobilised on a carbon electrode is described for the first time. The study of the effect of pH on the distribution of redox states demonstrates the existence of a pH equilibrium between the Ni-C and the Ni-L states. It is shown that the active site responds to the pH of the external solution, and that the increase in pH acts as a driving force that removes the proton further away from the active site. Studies under electrocatalytic conditions provides direct evidence of intermediates of the catalytic cycle. The role of Ni-SI, Ni-R, and Ni-C is confirmed. Furthermore, Ni-L is detected under turnover conditions and therefore shown to be an important intermediate in the cycle. The detection of different protonation states of Ni-L and Ni-R is proposed to provide information on the transport of the protons as they start to move away from the active site. In the investigation of O2 inhibition, Ni-B (detected spectroscopically) is directly related to the loss in activity upon the attack of O2 for the first time. Also, the formation of solely Ni-B from the reaction with O2 (no other O2-damaged species are detected) provides further evidence on the ability of O2-tolerant hydrogenases of having an effective mechanism for dealing with O2 tolerance. A thorough study on the interaction of CO with Hyd-1 proves unequivocally that this O2-tolerant hydrogenase does bind CO, and that CO does inhibit its catalytic activity (both H2 and H+ reduction). This helps clarify how CO interacts with O2-tolerant hydrogenases. Overall, the work in this thesis contributed to the understanding of key mechanistic aspects in O2-tolerant hydrogenases. The technique for combining protein film electrochemistry with IR spectroscopy in situ shall provide valuable opportunities for providing new insight into the mechanisms of hydrogenases and other metalloenzymes that bind small molecules.
Supervisor: Vincent, Kylie A. Sponsor: Universidad de Costa Rica ; Ministerio de Ciencia Y Tecnologia de Costa Rica
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available