Characterisation of cytochrome c 6 from Chlorella fusca

Abstract

A c-type monohaem, cytochrome c6was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an apparent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 20.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-terminal sequence shows high identity with other green algae cytochromes c6. The mid-point redox potential is about +350 mV between pH 5 and 9. The ferric and ferrous forms, and their pH equilibria, have been studied using visible, CD and EPR spectroscopies. The visible spectrum of the reduced cytochrome c6is typical of a c-type haem protein, with maxima at 274 nm, 318 nm (δ-peak), 416 nm (γ-peak), 522 nm (β-peak), 552–553 nm (α-peak). A 690 nm band, characteristic of a haem Met-His axial coordination of the haem group, is present in the oxidized form. At high pH values (≥ 8), cytochrome c6undergoes 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-values at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert with a pKaof 4. CD spectrum of Chlorella fusca cytochrome c6shows that the proteins must be mainly built up by α-helices. Even though there are similarities between Chlorella fusca cytochrome c6and that isolated from Monoraphidium braunii, no cross-reactivity with the antibodies raised against the Chlorella fusca cytochrome has been detected for the protein from Monoraphidium braunii.