Abstract
Cytochrome b562 is a periplasmic Escherichia coli protein; previous work has shown that heme can be attached covalently in vivo as a consequence of introduction of one or two cysteines into the heme-binding pocket. A heterogeneous mixture of products was obtained, and it was not established whether the covalent bond formation was catalyzed or spontaneous. Here, we show that coexpression from plasmids of a variant of cytochrome b562 containing a CXXCH heme-binding motif with the E. coli cytochrome c maturation (Ccm) proteins results in an essentially homogeneous product that is a correctly matured c-type cytochrome. Formation of the holocytochrome was accompanied by substantial production of its apo form, in which, for the protein as isolated, there is a disulfide bond between the two cysteines in the CXXCH motif. Following addition of heme to reduced CXXCH apoprotein, spontaneous covalent addition of heme to polypeptide occurred in vitro. Strikingly, the spectral properties were very similar to those of the material obtained from cells in which presumed uncatalyzed addition of heme (i.e. in the absence of Ccm) had been observed. The major product from uncatalyzed heme attachment was an incorrectly matured cytochrome with the heme rotated by 180 degrees relative to its normal orientation. The contrast between Ccm-dependent and Ccm-independent covalent attachment of heme indicates that the Ccm apparatus presents heme to the protein only in the orientation that results in formation of the correct product and also that heme does not become covalently attached to the apocytochrome b562 CXXCH variant without being handled by the Ccm system in the periplasm. The CXXCH variant of cytochrome b562 was also expressed in E. coli strains deficient in the periplasmic reductant DsbD or oxidant DsbA. In the DsbA- strain under aerobic conditions, c-type cytochromes were made abundantly and correctly when the Ccm proteins were expressed. This contrasts with previous reports indicating that DsbA is essential for cytochrome c biogenesis in E. coli.
Highlights
Cytochrome b562 is a hemoprotein expressed in the periplasm of the Gram-negative bacterium Escherichia coli [1]
The Dsb and cytochrome c maturation (Ccm) systems are believed to work in series during cytochrome c biogenesis such that as the unfolded apocytochrome is exported to the periplasm, it is first oxidized by DsbA
The Ccm apparatus has been shown to process cytochromes c from diverse prokaryotic and eukaryotic origins (e.g. Refs. 17 and 19 –21). In light of these recent data, we have investigated the action of the Ccm system on the R98C/Y101C (CXXCH) variant of E. coli cytochrome b562
Summary
Cytochrome b562 is a hemoprotein expressed in the periplasm of the Gram-negative bacterium Escherichia coli [1]. The ␣-band of such a spectrum of the pyridine- and hydroxide-treated crude periplasmic extract was at 550 nm (Fig. 1, inset), the wavelength expected for covalent attachment of heme to protein through two thioether linkages to a CXXCH peptide motif, as found in a typical cytochrome c.
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