CHARACTERIZATION OF CYTOCHROME C(6) FROM CHLORELLA-FUSCA

A c-type monohaem, cytochrome c(6) was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an appa rent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 2 0.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-termina l sequence shows high identity with other green algae cytochromes c(6) . The mid-point redox potential is about +350 mV between pH 5 and 9. T he ferric and ferrous forms, and their pH equilibria, have been studie d using visible, CD and EPR spectroscopies. The visible spectrum of th e reduced cytochrome c(6) is typical of a c-type haem protein, with ma xima at 274 nm, 318 nm (delta-peak), 416 nm (gamma-peak), 522 nm (beta -peak), 552-553 nm (alpha-peak). A 690 nm band, characteristic of a ha em Met-His axial coordination of the haem,group, is present in the oxi dized form. At high pH values (greater than or equal to 8), cytochrome c(6) undergoes an alkaline transition, with a pKa of 8.7. Between pH 3 and 9 the EPR spectrum is dominated by two rhombic species, with g-v alues at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert wit h a pK(a) of 4. CD spectrum of Chlorella fusca cytochrome c(6) shows t hat the proteins must be mainly built up by alpha-helices. Even though there are similarities between Chlorella fusca cytochrome c(6) and th at isolated from Monoraphidium braunii, no cross-reactivity with the a ntibodies raised against the Chlorella fusca cytochrome has been detec ted for the protein from Monoraphidium braunii.