CHARACTERIZATION OF CYTOCHROME-C(6) FROM THE CYANOBACTERIUM ANABAENA PCC-7119

A soluble monoheme c-type cytochrome c(6) has been isolated from the c yanobacterium Anabaena PCC 7119. It is a basic protein, with a molecul ar mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin i n the ferredoxin-NADP(+) reductase-dependent NADPH cytochrome c reduct ase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with on ly 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c(6) cytochromes from the green algae Chlorella fusca and Monoraphidi um braunii are also shown to accept electrons from Anabaena ferredoxin . The reduction potential of cytochrome c(6) at pH 6.7 was determined to be 338 mV and is pH dependent, with pK(a)(ox)=8.4+/-0.1 and pK(a)(r ed)approximate to 9.5. The ferric and ferrous cytochrome forms and the ir pH equilibria have been studied using visible, EPR and H-1-NMR spec troscopies. The amino acid sequence and the visible and NMR spectrosco pic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5-11. However, the EPR data for the ferri cytochrome are complex and show that in this pH range five distinct fo rms are present. Between pH 5 and 9 the spectrum is dominated by two r hombic species, with g-values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1 .56, which interconvert with a pK(a) of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which co incides with that determined from the pH dependence of the reduction p otential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectr al data, although having crystal field parameters atypical for this ty pe of coordination. Anabaena cytochrome c(6) is one more example of a heme protein for which the widely used crystal field analysis of the E PR data (truth diagram) fails to unequivocally determine the type of h eme-iron ligation.