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Title: Voltammetric comparisons of structurally homologous nitrite reducing cytochromes from Thioalkalivibrio nitratireducens and Escherichia coli
Author: Doyle, Rose-Marie
ISNI:       0000 0004 2735 241X
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2011
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The reduction of nitrite to ammonia by the pentaheme cytochrome c nitrite reductase, NrfA, has been well studied. The NrfAs are catalytically promiscuous enzymes that also reduce hydroxylamine and nitric oxide to ammonia and sulphite to sulphide. There are structural differences between NrfA enzymes from different organisms and the evolutionary related group of catalytic multi-heme cytochromes, which appear to have different catalytic and functional specificities. It is of interest to determine which structural features are important for effective catalysis and if the differences in the structure of these enzymes are indicative of evolved specificity or function. Here nitrite reduction by the octaheme cytochrome c nitrite reductase from the obligately haloalkaliphilic Thioalkalivibrio nitratireducens, TvNir, was compared to nitrite reduction by Escherichia coli NrfA using protein film electrochemistry. The kinetics of nitrite reduction by each enzyme were distinctive. The KM of TvNir decreased by ca. 500 uM as the pH was increased from pH 5 to pH 10, whereas contrastingly the KM of NrfA increased by less than 100 ~M from pH 4 to pH 9. Below pH 7 nitrite reduction by TvNir was substrate inhibited and this has never been detected for NrfA. Approximately 30% of the TvNir sample required reductive activation, whereas samples of purified NrfA are already catalytically competent. The possible origins of these differences in the sequence and structure of NrfA and TvNir are discussed. Spectropotentiometry and protein film voltammetry defined the reduction potentials of NrfB, part of the redox pathway to NrfA. The reduction potentials of NrfA and NrfB are very similar to those of TvNir and so the thermodynamic pathways allowing electron transfer to their active sites, enabling catalysis by these nitrite reductases are similar. The results of the protein film electrochemistry experiments to assess nitrite reduction by NrfAB were ambiguous and the further experiments required to address this are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available