Abstract
The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c2 by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c2 and c1, and class II c') and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nM to μM KD values). When present, heme induced conformational changes in class I apocytochromes (e.g. c2) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c2 and that heme converts apocytochrome c2 into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c2) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.
Highlights
Cytochrome c maturation (Ccm) forms thioether bonds between heme b and c-type apocytochromes
We chose in addition to apocytochrome c2 the class I membraneanchored cytochrome c1, for which maturation is independent of the CcmI-2 domain of CcmI, and the class II-soluble cytochrome cЈ, which has a nonglobular three-dimensional structure (Fig. 2A)
The secondary structure changes were monitored after incubating CcmI and apocytochrome c2 in the presence or absence of hemin, and the circular dichroism (CD) spectra obtained were compared with the sums of the spectra of the individual proteins recorded under the same experimental conditions (Fig. 7B)
Summary
Cytochrome c maturation (Ccm) forms thioether bonds between heme b and c-type apocytochromes. The c-type cytochromes are electron transfer proteins involved in energy transduction They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. The c-type cytochromes are ubiquitous electron transfer proteins involved in energy transduction in almost all living cells, and they play critical roles in other cellular pathways (e.g. apoptosis in eukaryotes) [1,2,3] These proteins always contain at least one conserved heme-binding site (C1XXC2H), where heme b (protoporphyrin IX-Fe) is covalently ligated. Heme modulates these binding interactions significantly, suggesting that CcmI holds the c-type apocytochromes tightly until their intermediate b-type derivatives are formed We propose that these intermediates are subsequently converted into mature c-type cytochromes upon completion of covalent heme ligation by the remaining components of the Ccm complex
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