An Engineered Cytochrome b 6 c 1 Complex with a Split Cytochrome b Is Able To Support Photosynthetic Growth of Rhodobacter capsulatus

Abstract

ABSTRACT The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc 1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b , and cytochrome c 1 subunits encoded by petA ( fbcF ), petB ( fbcB ), and petC ( fbcC ) genes organized as an operon. In the work reported here, petB ( fbcB ) was split genetically into two cistrons, petB6 and petBIV , which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b , respectively. These polypeptides resembled the cytochrome b 6 and su IV subunits of chloroplast cytochrome b 6 f complexes, and together with the unmodified subunits of the cytochrome bc 1 complex, they formed a novel enzyme, named cytochrome b 6 c 1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus . Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV , was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b 6 c 1 complex were similar to those of the cytochrome bc 1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc 1 complex, including antimycin A, and the redox properties of its b - and c -type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome b L heme was modified in a way reminiscent of that of a cytochrome b 6 f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280–16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc 1 complexes and the chloroplast cytochrome b 6 f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.