Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus

Abstract

c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys-Xaa-Xaa-Cys-His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b(562) containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b(562) to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CX(n)CH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.

Aberrant Attachment of Heme to Cytochrome by the Ccm System Results in a Cysteine Persulfide Linkage

“…13 cb 562 sequences with X 2 , X 3 , or X 4 are clearly substrates of Ccm and yield c-type cytochromes with the expected heme attachment. 11 Expression of sequences with X 5 or X 6 yields mature c-type cytochromes with heme attached and spectral properties consistent with the normal mode of heme attachment. 11 However, electrospray ionization (ESI) mass spectrometry (MS) of these purified proteins shows that they are actually mixtures containing, in addition to the protein of expected mass, very closely related species with extra mass relative to the expected mass of the apoproteins plus attached iron protoporphyrin IX.…”

“…8,10 Variants containing different lengths of intervening sequence between the cysteines (CX n CH, where n ) 3, 4, 5, or 6) have recently been generated. 11 Sequences with X 3 and X 4 are known in natural cytochromes, 12 but there are no natural examples with X 1 , X 5 , or X 6 . 13 cb 562 sequences with X 2 , X 3 , or X 4 are clearly substrates of Ccm and yield c-type cytochromes with the expected heme attachment.…”

“…For X 5 and X 6 , these proteins of higher mass have been shown to be products of the Ccm machinery. 11 Given their abundance, we reasoned that understanding their origin may shed light upon the mysterious mechanism of the Ccm enzymatic apparatus. In this work, we have identified the location and elemental origin of the extra 32 Da in each protein using high-resolution accurate ion-trap MS combined with peptide fragmentation.…”

The broad diversity of heme-protein cross-links: An overview

“…Due to the well-folded structure of four-helix bundle and enhanced stability with respect to cyt b 562 , cyt cb 562 was extensively used by Tezcan and co-workers [28][29][30] to create novel biomaterials, such as one-dimensional nanotubes and two-or three-dimensional crystalline arrays, by rational design of metal-binding sites on protein surface to introduce protein self-assembly. Moreover, when a hemebinding motif of CX 3 CH or CX 4 CH was constructed in cyt b 562 , cyt cb 562 could also be maturated with heme attached correctly through two thioether bonds, indicating these motifs are the substrate of cyt c maturation (Ccm) apparatus [31]. Meanwhile, with an unnatural heme-binding motif of CX n CH (n = 1, 5, or 6), an extra sulfur was incorporated into one thioether bond of the maturated cyt cb 562 , resulting in formation of an aberrant persulfide linkage [32].…”

Molecular Basis Behind Inability of Mitochondrial Holocytochrome c Synthase to Mature Bacterial Cytochromes

“…Although the bacterial CCM system (i.e. System I) can successfully mature a wide range of mono-and multiheme cytochromes, containing simply a CX n CH motif (where X n ϭ 1-4 intervening residues) (21,24,42,43), additional structural features of eukaryotic cytochromes contribute to the strict substrate specificity of HCCS. For example, previous genetic studies have suggested that the conserved phenylalanine residue in cyt c ␣ helix-1 is critical for HCCS maturation by serving as a determinant for substrate recognition (step 2) because substitution of this residue can result in poor cyt c yields (21, 25) (Phe 11 in human cyt c; the equivalent of this position in bacterial cytochromes is not fully conserved (21, 44)).…”