Chapter 8 Conclusions and future research perspectives
The aims of the present work were two-fold: first, to clone and sequence the nirS gene from T. pantotropha, so as to be able to compare cytochrome cd\(_1\) nitrite reductase from this organism with the enzyme from other sources, and second, to investigate systems by which the nirS gene could be expressed so as to yield a holoprotein containing both the c- and the d\(_1\) haems. The ultimate goal of this work, which is beyond the scope of the present project, is to construct a number of site-directed mutants of cytochrome cd\(_1\) in which the key residues, such as the haem ligands and the histidines at the active site, are altered. This would allow the role of these residues to be more clearly defined.
In Chapter 3, the sequence of the nirS gene and its product, cytochrome cd\(_1\) was reported. Knowledge of the cytochrome cd\(_1\) protein sequence has allowed the crystal structure to be refined to 1.28 \(\unicode{x212b}\) and has confirmed the haem ligands in the oxidised form (His-17 and His-69 at the c-haem, Tyr-25 and His-200 at the d\(_1\) haem). The sequence also confirms that eight N-terminal residues are not visible in the crystal structure because of disorder in the crystal packing. The major finding in Chapter 3 was that the haem ligands His-17 and Tyr-25 are not conserved in the cytochrome cd\(_1\) family. This was a surprising finding in view of the overall high similarity of the protein sequences and some implications for the catalytic mechanism of the enzyme were discussed. It was noted that structural data for cytochrome cd\(_1\) from other organisms would be a valuable addition to the current data. Very recently, the structure of cytochrome cd\(_1\) in the oxidised state from Ps. aeruginosa has been solved [354]. Although the structure itself has not yet been published, some of the details have been made available. The c-haem is ligated by a methionine and a histidine residue, as was predicted from MCD spectroscopy for cytochrome cd\(_1\) both from Ps. aeruginosa [124] and from Ps. stutzeri Zobell [125]. The d\(_1\) haem is ligated by histidine and by a hydroxide ion. Interestingly, Ps. aeruginosa cytochrome cd\(_1\) is the only other example, apart from that of T. pantotropha cytochrome cd\(_1\), of a cytochrome cd\(_1\) with a tyrosine residue close to the N-terminus of the protein. In the case of Ps. aeruginosa this tyrosine, Tyr-10, does not act directly as a ligand to the d\(_1\) haem, but is instead hydrogen-bonded to the hydroxide ion which in turn is the sixth ligand to the d\(_1\) haem. Furthermore, replacement of Tyr-10 with phenylalanine has no effect on the catalytic activity, optical spectroscopy or electron transfer kinetics of Ps. aeruginosa cytochrome cd\(_1\) [354]. These data strengthen the case for significant structural and mechanistic differences between the cytochromes cd\(_1\), but also emphasise the importance of assessing the role of Tyr-25 in the T. pantotropha enzyme using as many approaches as possible.
Chapters 4 to 6 of the present work were concerned with the expression of the cloned nirS gene in different systems. In Chapter 4 it was shown that the T. pantotropha nirS gene could be expressed in E. coli to give a semi-apo protein containing only c-type haem. It was then possible to reconstitute the recombinant protein with purified d\(_1\) haem, producing a protein with some of the spectroscopic and catalytic features of the native enzyme. This approach will be of use in the production of site-directed mutants that are structurally unstable or enzymically inactive when expressed as the holoprotein. Such mutants would not support the growth of the host organism under denitrifying conditions and so little or no protein could be recovered for further studies. However, provided that the semi-apo form of the mutant protein was stably maintained in E. coli, it should be possible to reconstitute the recombinant protein as described. Future work in this area will require improvements in the yield of recombinant protein and the optimisation of the reconstitution protocol.
Chapter 5 described the expression of the T. pantotropha nirS gene in Ps. aeruginosa. In this case, it was hoped that the recombinant protein would be able to sequestrate the d\(_1\) haem that was synthesised by the host organism, so as to yield the active holoenzyme. However, some difficulties were experienced both with the solubility of the recombinant enzyme during anaerobic growth and with the DNA construct used. It appeared that the presence of either the regulatory region upstream of the cloned nirS gene or that of the truncated downstream nirE gene had an adverse effect on the growth of the host cells. In future work using any heterologous host organism, a DNA construct containing only the structural gene, nirS, is recommended. Nevertheless, recombinant cytochrome cd\(_1\) with some of the spectroscopic and catalytic properties of the native enzyme was produced, and this system could be of use for identifying mutants of cytochrome cd\(_1\) in which enzymatic activity has been completely abolished.
Chapter 6 detailed the construction of a nirS deletion mutant in T. pantotropha and its use as a host for expression of the cloned nirS gene. It was shown that the mutant could be complemented with the wild-type gene on a low copy number plasmid, fully restoring anaerobic growth on nitrate with no deleterious effects. However, expression of a cytochrome cd\(_1\) mutant in which the d\(_1\) haem ligand, Tyr-25, was replaced with phenylalanine did not restore growth to the deletion mutant strain; furthermore, the amount of expressed enzyme was extremely low, indicating that it may have been degraded. These data indicate that Tyr-25 is an essential residue for the function of T. pantotropha cytochrome cd\(_1\), although strictly, as the recombinant protein could not be isolated, it was not possible to determine whether Tyr-25 is essential for catalytic activity. However, this interpretation of the data is very plausible; it may be that as the cells are incapable of nitrite reduction, they are placed under stress because of the accumulation of nitrite and the inactive cytochrome cd\(_1\) is degraded as part of a general stress response. The presence of elevated levels of pseudoazurin, cytochrome c peroxidase and a previously unidentified c-type cytochrome may also all be indications of a stress response. These data illustrate the problem of expressing a cytochrome cd\(_1\) mutant that cannot support the growth of the host organism under denitrifying conditions. Possible solutions to this problem may include (1) the co-expression of a copper nitrite reductase in the deletion mutant strain, which would allow denitrifying growth and also the isolation of the mutant cytochrome cd\(_1\), and (2) investigation of the expression of cytochrome cd\(_1\) mutants under different growth conditions. So far is as known, the genes for d\(_1\) biosynthesis, as well as the nirS gene, are activated during anaerobic, denitrifying growth, but further understanding of the regulation of denitrification may reveal a way in which aerobic expression of these genes can be achieved.
Finally, in Chapter 7, the transcription of the nirS gene was explored. It was shown that the nirS gene was transcribed as a monocistronic mRNA. The transcription start site of the gene was also identified. The former result was of some importance, as it established that the nirS deletion described in Chapter 6 could be constructed without disruption of the d\(_1\) biosynthesis genes situated downstream of the nirS gene. Additionally, an analysis of some of the denitrification gene sequences that are currently known indicates a common regulatory mechanism and strongly suggests that these genes are transcribed using a specific sigma factor. However, it is abundantly clear that at present, very little is understood of the transcriptional regulation of denitrification genes, and a number of future experiments in this direction were proposed at the end of Chapter 7. Elucidating the regulation and function of the many denitrification genes that have been identified looks certain to be the major challenge in the field of denitrification research in future years.