Cytochrome c(y) of Rhodobacter capsulatus is attached to the cytoplasmic membrane by an uncleaved signal sequence-like anchor

During the photosynthetic growth of Rhodobacter capsulatus, electrons are conveyed from the cytochrome (cyt) bc(1) complex to the photochemical reaction center by either the periplasmic cyt c(2) or the membrane-bound cyt c(y). Cyt c(y) is a member of a recently established subclass of bipartite c-type cytochromes consisting of an amino (N)-terminal domain functioning as a membrane anchor and a carboxyl (C)-terminal domain homologous to cyt c of various sources, Structural homologs of cyt c(y) have now been found in several bacterial species, including Rhodobacter sphaeroides. In this work, a C-terminally epitope-tagged and functional derivative of R. capsulatus cyt c(y) was purified from intracytoplasmic membranes to homogeneity. Analyses of isolated cyt c(y) indicated that its spectral and thermodynamic properties are very similar to those of other c-type cytochromes, in particular to those from bacterial and plant mitochondrial sources, Amino acid sequence determination for purified cyt c(y) revealed that its signal sequence-like N-terminal portion is uncleaved; hence, it is anchored to the membrane, To demonstrate that the N-terminal domain of cyt c(y) is indeed its membrane anchor, this sequence was fused to the N terminus of cyt c(2). The resulting hybrid cyt c (MA-c(2)) remained membrane bound and was able to support photosynthetic growth of R. capsulafin in the absence of the cyt c(y) and c(2). Therefore, cyt c(2) can support cyclic electron transfer during photosynthetic growth in either a freely difusible or a membraneanchored form, These findings should now allow for the first time the comparison of electron transfer properties of a given electron carrier when it is anchored to the membrane or is freely diffusible in the periplasm.