Two Unusual Amino Acid Substitutions in Cytochrome b of the Colorless AlgaPolytomellaspp.: Correlation with the Atypical Spectral Properties of the bHHeme

The dithionite-reduced spectra of the purified bc1complexes from the colorless algaPolytomellaspp. and the closely related green algaChlamydomonas reinhardtiiwere compared. The spectrum of the bc1complex fromC. reinhardtiishowed a profile similar to those of the bc1complexes from other species. In contrast, the bc1complex fromPolytomellaspp. exhibits a double-peak spectrum in the α-band region, where the absorption bands of cytochrome c1and cytochrome b are completely resolved. To further understand the molecular basis of these spectroscopic differences, the mitochondrial gene encoding cytochrome b ofPolytomellaspp. was cloned, sequenced, and compared with that ofC. reinhardtii.ThePolytomellaspp. cytochrome b gene is 1113 bp long and does not contain introns. The deduced protein sequence exhibits 56% identity and 68% similarity with the cytochrome b ofC. reinhardtii,and in a phylogenetic analysis it clearly affiliated with the b-type cytochromes ofC. reinhardtiiandC. smithii.A comparison of the primary sequences of thePolytomellaspp. cytochrome b with other b-type cytochromes, and its analysis based on the structure featuring eight transmembrane stretches, allowed the identification of a tyrosine in position 114, which substitutes for a tryptophan present in all mitochondrial b-type cytochromes sequenced to date. In addition, the primary sequence of the cytochrome b fromPolytomellaspp. has a serine at position 36, instead of a nonpolar residue (alanine or leucine) found in all other species. In the proposed model for cytochrome b, both residues Tyr114and Ser36are in close proximity to the high-potential bHheme. The above data suggest that the polar residues Y114and S36, each one by itself or in combination, may interact with heme bHofPolytomellaspp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochromeb.