Molecular genetic manipulation and characterization of the high-potential chain of cyclic electron transport in Rhodobacter sphaeroides

Abstract

The Rieske iron-sulfur subunit of the cytochrome bc\sb{1} complex of Rhodobacter sphaeroides assembles in the E. coli and R. sphaerodies membranes with its iron-sulfur cluster intact, in the absence of the cytochrome subunits. This assembly, in light of predicted structural features of the hydrophobic amino-terminus and proteolytic release of a soluble domain, suggests the subunit may be anchored to the membrane by a single amino-terminal, transmembrane helix. Sequence homology with dioxygenase subunits binding a spectrally similar 2FE-2S cluster, suggests the fully conserved residues of the cytochrome bc\sb{1} and b\sb{6}f sequences ligating the iron-sulfur cluster may be the first and third cysteines and both histidines. When Gly133, neighboring the ligands of the cluster, is replaced by aspartate, quinol oxidation is specifically impaired at the Q\sb{\rm o} site.The heme-binding domain of cytochrome c\sb{1} released from the membrane to the periplasmic space by genetic removal of the carboxyl-terminal anchor is capable of reducing cytochrome c\sb{2} at a near normal rate. Truncation of cytochrome c\sb{1} causes a drop in its midpoint potential of 40 mV. The crippled complex remaining in the membrane lacking both the low potential b heme and cytochrome c\sb{1} can still bind stigmatellin and antimycin.Electron transport assay of a R. sphaeroides strain with cytochrome c\sb{2} genetically deleted shows that cytochrome c\sb{2} is essential to connect the cytochrome bc\sb{1} complex with the reaction center. A soluble c cytochrome, induced by second site mutations in the mutant, restores electron transfer between the cytochrome bc\sb1 complex and the reaction center.The usefulness of the alkaline phosphatase gene fusion assay of membrane protein topology is confirmed using the Rhodobacter sphaeroides reaction center subunit L. The fusions with junctions expected to lie near the periplasmic surface, based on the crystallographic structure of the reaction center, have high activity. These results suggest the assay can be applied to some membrane proteins foreign to E. coli.U of I OnlyETDs are only available to UIUC Users without author permissio