Structural and biochemical analysis of two copper proteins - plastocyanin from spinach and nitrite reductase from Rhodobacter sphaeroides 2.4.3

Doctoral thesis, 2006

Plastocyanin from spinach and nitrite reductase from Rhodobacter sphaeroides 2.4.3 are two copper-containing proteins that are involved in biological electron transport chains: plastocyanin operates in photosynthesis and nitrite reductase takes part in the denitrification pathway. X-ray crystallography together with biochemical analysis of both macromolecules has provided us with new insight into the chemical details of their biological mechanisms. Mutational studies of plastocyanin were performed to elucidate the importance of a hydrophobic patch for electron transfer activity. In addition, investigation of the thermostability of plastocyanin by the insertion of a disulfide bond indicated an increased stability of the type 1 Cu site in one mutant and resulted in changes in crystallisation properties of the protein. The crystal structures of the reduced and oxidised species of nitrite reductase were solved and compared with regard to Cu site environment. Further crystallographic work includes structures of nitrite reductase at pH 8.4 and pH 6.0 and with or without nitrite. These structures were also compared with respect to Cu site geometry, and were discussed in light of the pH dependence of enzyme activity, substrate affinity and gene expression.