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Mechanistic Insights Into Arsenite Oxidase and Implications for Its Use As a Biosensor

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Mechanistic Insights Into Arsenite Oxidase and Implications for Its Use As a Biosensor Mechanistic insights into arsenite oxidase and implications for its use as a biosensor Cameron Misha Manson Watson UCL Submitted for the degree of Doctor of Philosophy 1 I, Cameron Misha Manson Watson, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed: Dated: 2 Abstract Arsenic is an environmental toxin which poses a threat to >140 million people worldwide. The respiratory enzyme arsenite oxidase (Aio) from various bacteria couples the oxidation of arsenite to the reduction of electron acceptors. The Aio from Rhizobium sp. str. NT-26 is in development as an arsenic biosensor. Aio consists of a large subunit (AioA), containing a molybdenum centre and a 3Fe-4S cluster, and a small subunit (AioB) containing a Rieske 2Fe-2S cluster. The first objective was to identify the rate-limiting step of Aio catalysis to establish if the rate could be improved. The rate-limiting step was found to be electron transfer from the 2Fe-2S cluster to cytochrome c by using stopped-flow spectroscopy, steady- state kinetics and isothermal titration calorimetry. An AioB mutant (F108A) specifically reduced activity with cytochrome c by affecting electron transfer. The AioB subunit was expressed alone and was able to weakly associate with cytochrome c suggesting that the AioA subunit is important in the cytochrome c interaction. Unfortunately, the AioA subunit was unstable alone so its cytochrome c interaction was not characterised. Most AioB possess a disulphide bridge proposed to be involved in electron acceptor selectivity. The NT-26 Aio does not possess a disulphide bridge while that of Alcaligenes faecalis does. Site-directed mutagenesis introduced and removed a disulphide bridge into the NT-26 and Alcaligenes faecalis Aio respectively. Presence of the disulphide bridge increased activity with azurin and decreased activity with cytochrome c. The oxidation of antimonite by Aio was examined to determine how the presence of antimony might affect biosensor performance and to assess if Aio could be used as an antimonite biosensor. Antimonite was found to be a potent, competitive inhibitor of Aio because the product of antimonite oxidation dissociates slowly from the active site. The impact of this on the biosensor’s viability is discussed. 3 Impact Statement The rate-limiting step of Aio catalysis has been shown to be electron transfer to the electron acceptor, cytochrome c, and this work has been published. Cytochrome c serves as a mediator between the Aio and the electrode of the arsenic biosensor. It was found that it is unlikely that this interaction can be improved and that only a 50% increase could be achieved before further engineering of the enzyme would be required meaning that the wild-type Aio should be used for the biosensor. The role of the AioB disulphide bridge in electron acceptor specificity was explored. The AioB disulphide bridge has previously been suggested to be involved in electron acceptor specificity and the data presented in this thesis provides experimental evidence confirming it which is expected to be published. The expression system developed for AioB developed could also be useful for studies into Rieske function. The phylogeny of the Rieske proteins suggests that the disulphide bridge was substituted by a hydrophobic, aromatic residue in the Aio of the Alphaproteobacteria, possibly to facilitate electron transfer to cytochrome c. The Rieske protein of the bc1 complex uses cytochrome c1 as an electron acceptor but possesses a disulphide bridge. The results of this study could be used to inform mutagenesis and engineering studies with the bc1 complex. A heterologous expression system for the Pseudomonas aeruginosa azurin has been developed. Azurin has been shown to have potential clinical significance in the treatment of malaria, HIV and some cancers. Azurin is expensive to purchase and the expression system described in this thesis could facilitate further research into its clinical potential or marketisation by providing a cheaper source of the protein. The NT-26 Aio has been shown to be able to oxidise antimonite and this process was characterised in this thesis. This is the first study into the interaction between a molybdoenzyme and antimony and is expected to be published. It was found that the previously reported kinetics values for antimonite oxidation were inaccurate and corrected values have been presented. Antimonite was found to act as a potent inhibitor of arsenite oxidation by the Aio and the mechanism for this was 4 characterised, demonstrating that the oxidised antimony product dissociates slowly from the active site, retarding arsenite oxidation. This finding demonstrates that 1) the NT-26 Aio is inappropriate as a biosensor for antimonite as the rate of catalysis is too slow; and 2) that the Aio may not be able to be used as a biosensor in waters with high antimony concentrations such as in industrial waste or fracking sites. A market analysis is presented and discusses the benefits of the Asian versus North American markets. Considering the findings of this thesis, the Asian market is considerably more desirable than the North American as antimony pollution appears to be considerably lower. 5 Table of Contents Abstract 3 Impact Statement 4 Acknowledgements 20 Abbreviations 21 1.1 Arsenic and antimony 24 1.1.1 Arsenic 24 1.1.2 Antimony 28 1.1.3 Arsenic and antimony in biology 30 1.2 Molybdenum enzymes 34 1.2.1 Xanthine Oxidase family 35 1.2.2 Sulphite oxidase family 36 1.2.3 Mo/W-bis pterin guanosine dinucleotide family 37 1.2.4 Role of pyranopterins 38 1.3 Rieske proteins 39 1.3.1 Rieske families 40 1.3.2 Rieske Evolution 43 1.4 Arsenite oxidase 43 1.4.1 The Aio enzyme subunits and cofactors 45 1.4.2 Evolution of arsenite oxidase 47 1.4.3 The arsenite oxidase gene cluster 49 1.4.4 Electron acceptors to the arsenite oxidase 50 1.4.5 Catalytic mechanism of the arsenite oxidase 52 1.4.6 Kinetics of Aio 54 1.5 Biosensors 56 6 1.5.1 Arsenic Biosensors 58 1.5.2 Business case for Aio as an arsenic biosensor 61 1.6 Protein engineering 63 1.6.1 Rational Design 63 1.6.2 Directed Evolution 64 1.7 Aims of this study 65 2.1 Introduction 67 2.1.1 Electron transport in arsenite oxidase 67 2.1.2 Stopped-flow spectroscopy 69 2.1.3 Isothermal titration calorimetry 70 2.1.4 Aims 72 2.2 Methods 73 2.2.1 WT and F108A Aio expression systems 73 2.2.2 Conformation of presence of aioBA genes in recombinant plasmids by restriction digestion and sequencing 73 2.2.3 Cloning of the aioB gene for heterologous expression in E. coli 74 2.2.4 Aio expression and purification 74 2.2.5 Expression and purification of AioB 75 2.2.6 Protein concentration determination 76 2.2.7 UV-visible spectroscopy 76 2.2.8 Inductively coupled plasma mass spectrometry 76 2.2.9 Polyacrylamide gel electrophoresis 77 2.2.10 Steady-state kinetics 77 2.2.11 The oxidised and reduced spectra of horse heart cytochrome c 78 2.2.12 Stopped-flow spectroscopy of Aio with arsenite and cytochrome c 79 7 2.2.13 Stopped flow kinetics of AioB versus cytochrome c 80 2.2.14 Isothermal titration calorimetry of Aio versus cytochrome c 80 2.2.15 ITC of AioB versus cytochrome c 81 2.2.16 Statistical Analysis 82 2.3 Results and Discussion 83 2.3.1 Confirmation of the recombinant plasmids containing the aioBA genes 83 2.3.2 Purification and co-factor determination of the WT and F108A Aio 84 2.3.3 The visible absorbance spectra of WT Aio 85 2.3.4 Arsenite steady-state kinetics of WT Aio with DCPIP and cytochrome c 86 2.3.5 The reductive-half reaction of Aio 92 2.3.6 The oxidative-half reaction of Aio 95 2.3.7 The interaction of the Aio with cytochrome c 96 2.3.8 The mechanism of Aio catalysis 100 2.3.9 The effect of the AioB-F108A mutation on Aio Activity 103 2.3.10 The effect of the F108A mutation on electron transfer rates 106 2.3.11 The role of the AioB-F108 residue in electron transfer 109 2.3.12 Investigation into the interaction of AioB with horse heart cytochrome c 112 2.3.13 Cloning of the aioB gene 113 2.3.14 Expression and characterisation of heterologously expressed AioB 114 2.3.15 The interaction of AioB with cytochrome c 117 2.3.16 AioB heterologous expression system in E. coli 121 2.3.17 Engineering higher catalytic rates for Aio 122 2.3.18 Conclusions 123 8 3.1 Introduction 126 3.1.1 Structural diversity in arsenite oxidase Rieske proteins 127 3.1.2 Electron acceptor specificity of arsenite oxidases 128 3.1.3 Aims 129 3.2 Methods 131 3.2.1 Conformation of expression systems by restriction digest and sequencing 131 3.2.2 Azurin expression and purification 131 3.2.3 Aio expression and purification 132 3.2.4 Concentration determination 132 3.2.5 Characterisation of enzyme preparation purity, co-factor content and UV-visible spectra 133 3.2.6 Structural Alignment and electrostatic surface generation 133 3.2.7 Steady-state Kinetics 133 3.2.8 Statistical Analysis 135 3.2.9 Sequence alignment and phylogenetic tree reconstruction 135 3.3 Results 136 3.3.1 Expression and purification of azurin 136 3.3.2 Characterisation of heterologously expressed Pseudomonas aeruginosa azurin 137 3.3.3 Expression of WT and mutant arsenite oxidase from NT-26 and A.
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