N-15-labelling and preliminary heteronuclear NMR study of Desulfovibrio vulgaris Hildenborough cytochrome c(553)

When using heteronuclear NMR, N-15-labelling is necessary for structural an alysis, dynamic studies and determination of complex formation. The problem s that arise with isotopic labelling of metalloproteins are due to their co mplex maturation process, which involves a large number of factors. Cytochr omes c are poorly expressed in Escherichia coli and the overexpression that is necessary for N-15-labelling, requires an investigation of the expressi on host and special attention to growth conditions. We have succeeded in th e heterologous expression and the complete and uniform isotopic N-15-labell ing of the cytochrome c(553) from Desulfovibrio vulgaris Hildenborough, in a sulphate-reducing bacterium, D. desulfuricans G200, by using a growth med ium combining N-15-ammonium chloride and N-15-Celtone. These conditions all owed us to obtain approximate to 0.8 mg.L-1 of pure labelled cytochrome c(5 53). H-1 and N-15-assignments for both the oxidized and the reduced states of cytochrome c(553) were obtained from two-dimensional heteronuclear exper iments. Pseudocontact effects due to the haem Fe3+ have been analysed for t he first time through N-15 and H-1 chemical shifts in a c-type cytochrome.