Assembly of the Rieske Iron-Sulfur Subunit of the Cytochrome bc1 Complex in the Escherichia coli and Rhodobacter sphaeroides Membranes Independent of the Cytochrome b and c1 Subunits

Steven R. Van Doren, Chang Hyon Yun, Antony R. Crofts, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

Abstract

The Rieske iron-sulfur subunit of the cytochrome bc1 complex from Rhodobacter sphaeroides has been expressed in Escherichia coli and also in a strain of Rb. sphaeroides lacking the other subunits of the bc1 complex. PCR products encoding the full-length subunit were introduced into expression vectors to produce the subunit alone or the subunit fused behind the mature portion of the E. coli maltose binding protein (MBP), but lacking the MBP signal sequence. These proteins are both located in the cytoplasmic membrane. The unfused Rieske subunit assembles a Rieske-like iron-sulfur cluster, but with EPR characteristics which differ from the normal rhombic signal observed in the cytochrome bc1 complex. The overproduced MBP fusion protein, on the other hand, does not contain an EPR-detectable iron-sulfur cluster. Subfragments of the Rieske subunit lacking the amino-terminal hydrophobic anchor also lack the iron-sulfur cluster were expressed in E. coli. When expressed in Rb. sphaeroides in the absence of the cytochrome b and c1 subunits, the fully metalated Rieske subunit with the diagnostic gy = 1.90 EPR signal is observed in the cytoplasmic membrane. The fact that the Rieske subunit has an assembled iron-sulfur cluster and is bound to either the E. coli or the Rb. sphaeroides membrane in the absence of the other subunits of the bc1 complex demonstrates a mode of membrane attachment independent of the other components of the complex. These data are consistent with models in which the Rieske subunit is bound to the membrane via a single membrane-spanning helix located near the amino terminus. Proteolytic cleavage at a site just downstream of the putative membrane anchor results in a water-soluble form of the Rieske subunit from Rb. sphaeroides, providing further support for this model.

Original languageEnglish (US)
Pages (from-to)628-636
Number of pages9
JournalBiochemistry
Volume32
Issue number2
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry

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