Studies of multiheme proteins from the disimilatory metal reducing bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens
Genomic analysis of the dissimilatory metal-reducing bacteria Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA has shown that both are capable of expressing an unprecedented number of multiheme cytochromes. The isolation and characterisation of two of the multiheme cytochromes from these bacteria is the subject of this thesis. Octaheme tetrathionate reductase (OTR) from Shewanella oneidensis has been previously shown to catalyse the reduction of tetrathionate to thiosulfate (Rothery, 2003). Despite having an amino-acid sequence suggesting the presence of eight standard CXXCH hemeattachment motifs, the crystal structure of OTR has shown the enzyme to contain seven normally-coordinated bis-histidine-ligated hemes, with one unusual lysine-coordinated heme at the active site. Analysis of the structure of this enzyme has shown its heme architecture to have a significant similarity to those of hydroxylamine oxidoreductase and the pentaheme cytochrome c nitrite reductases. An improved protocol for the purification of OTR has been developed, and work has been carried out in order to identify further possible substrates for the enzyme. Results show that OTR is capable of reducing nitrite, hydroxylamine, nitrous oxide and nitric oxide, suggesting that OTR may have a role as a nitrogen cycle enzyme. In particular, the reactions of OTR with hydroxylamine and nitrite have been shown to be more efficient than that with tetrathionate (kcat / KM = 5.3 x 105 M^-1 s^-1 and 3.9 x 105 M^-1 s^-1 respectively). The product of the reactions of nitrite and hydroxylamine with OTR has been directly detected in solution and shown to be the ammonium ion. This activity is consistent with a nitrogen cycle enzyme, with the conversion of nitrite to ammonium representing a “short cut” in the cycle, as performed by the cytochrome c nitrite reductases. In addition to this, site-directed mutagenesis has been used to investigate the catalytic properties of key active site residues, including the unusual heme-ligating lysine. The complete genome sequence of Geobacter sulfurreducens PCA was determined in 2003 and genomic analysis predicted the presence of a putative octaheme protein, GSU0357. This protein was predicted to contain an unusual CXXCK heme-binding amino acid motif and showed a high sequence similarity to the pentaheme cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774. The purification of GSU0357 has been non-trivial and a range of approaches have been used. The ability of GSU0357 to act as a nitrite reductase has been confirmed.