Two unusual amino acid substitutions in cytochrome b of the colorless alga Polytomella spp.: correlation with the atypical spectral properties of the bH heme

Abstract

The dithionite-reduced spectra of the purified bc1 complexes from the colorless alga Polytomella spp. and the closely related green alga Chlamydomonas reinhardtii were compared. The spectrum of the bc1 complex from C. reinhardtii showed a profile similar to those of the bc1 complexes from other species. In contrast, the bc1 complex from Polytomella spp. exhibits a double-peak spectrum in the alpha-band region, where the absorption bands of cytochrome c1 and cytochrome b are completely resolved. To further understand the molecular basis of these spectroscopic differences, the mitochondrial gene encoding cytochrome b of Polytomella spp. was cloned, sequenced, and compared with that of C. reinhardtii. The Polytomella spp. 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 of C. reinhardtii, and in a phylogenetic analysis it clearly affiliated with the b-type cytochromes of C. reinhardtii and C. smithii. A comparison of the primary sequences of the Polytomella spp. 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 from Polytomella spp. 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 Tyr114 and Ser36 are in close proximity to the high-potential bH heme. The above data suggest that the polar residues Y114 and S36, each one by itself or in combination, may interact with heme bH of Polytomella spp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochrome b.