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Title: Catalytic Properties of a Periplasmic Nitrate Reductase : A Voltammetric Perspective
Author: Gates, Andrew James
ISNI:       0000 0004 2672 5130
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2008
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In Paracoccus pantotrophus, periplasmic nitrate reduction is performed by NapAB. This dissimilatory system is implicated in maintaining membrane redox homeostasis by linking ubiquinol oxidation with the reduction of nitmte, during aerobic metabolism of highly reduced carbon sources. NapA contains a bis-molybdopterin guanine dinucleotide cofactor and an iron-sulphur cluster. The associated di-haem subunit, NapB mediates catalytic electron flow from the quinol-oxidase electron donor, Nape. Protein film voltammetry (PFV) revealed steady-state electrocatalysis from NapAB in the presence of nitrate. At pH 7, nitrate reduction is onset <0.15 V and increases across the potential mnge where ubiquinollevels predominate in vivo. Though NapAB cannot opemte upstream of cytochrome be], its opemting potential is entirely consistent with the proposed physiological role of this enzyme in productive redox balancing. The magnitude of the electrocatalytic current-potential profile was greatest at acidic pH (~7) and responded readily to micromolar concentmtions of nitrate. At pH 6 and 7, KM values of ca. 23 and 45 J.lM were defined at the activity peak potential and 40 V, respectively. These values are in good agreement with the KM value of <15 J.lM defined for NapAB in intact cells of P. pantotrophus M-6 and thus bring the KM value for this enzyme into line with those of related proteins. Electrocatalytic activity was further modulated below ca. -0.15 V, where attenuation was observed. Such 'tunnel-diode' behaviour was maintained at [S]»KM and the high potential wave flank showed greatest pH dependence. At pH 6, potentiometric titration confirmed that activity attenuation occurred in the vicinity of the [4Fe-4Sf+/I + redox couple. A competitive inhibitor, thiocyanate had little impact on wave shape. However, NapAB purified from bacteria grown in tungsten-supplemented media showed a similar waveform to the molybdoenzyme, but where catalytic onset was displaced to lower potentials, while the position of the low potential wave flank was unchanged. With activity attenuation attributed to a redox couple distinct from the active site, modelling indicates the M06 + 15 + and M05 +/ 4+ potentials likely lie in close proximity in the high potential wave flank, thus a semi-cooperative redox process may describe the redox chemistry of the molybdenum site, consistent with the presence of sub-stoichiometric M05+ populations in EPR experiments.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available