The substitution of alanines for the two cysteines which form thioether linkages to the haem group in cytochrome c 552 from Hydogenobacter thermophilus destabilises the native protein fold. The holo form of this variant slowly converts into a partially folded apo state that over prolonged periods of time aggregates into fibrillar structures. Characterisation of these structures by electron microscopy and thioflavin-T binding assays shows that they are amyloid fibrils. The data demonstrate that when the native state of this cytochrome is destabilised by loss of haem, even this highly K K-helical protein can form L L-sheet structures of the type most commonly associated with protein deposition diseases. ß

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A new approach was developed to overproduce 15 N-enriched yeast iso-1-cytochrome c in the periplasm of Escherichia coli in order to perform a study of the motions in the ms -ms time scale on the oxidized and reduced forms through rotating frame 15 N relaxation rates and proton/deuterium exchange studies. It is confirmed that the reduced protein is rather rigid whereas the oxidized species is more flexible. The regions of the protein that display increased internal mobility upon oxidation are easily identified by the number of residues experiencing conformational equilibria and by their exchange rates. These data complement the information already available in the literature and provide a comprehensive picture of the mobility in the protein. In particular, oxidation mobilizes the loop containing Met80 and, through specific contacts, affects the mobility of helix 3 and possibly of helix 5, and of a section of protein connecting the heme propionates to helix 2. The relevance of internal motions to molecular recognition and to the early steps of the unfolding process of the oxidized species is also discussed. In agreement with the reported data, subnanosecond mobility is found to be less informative than the ms -ms with respect to redox dependent properties.Keywords: cytochrome c; backbone dynamics; NMR relaxation.Structural differences between the native state of the oxidized and reduced mitochondrial cytochromes c have been revealed by analysis of their crystal and solution structures. These differences are small but significant and mainly involve the heme propionate-7 and surrounding residues [1 -6]. Such conformational differences may play a role both in the electron transfer process and in molecular recognition. Oxidized and reduced cytochrome c also have different stability towards unfolding agents, the reduced cytochrome c being significantly more stable than the oxidized protein towards unfolding [7,8]. The difference in stability of the two redox forms has been tentatively related to different protein flexibilities. This hypothesis was based mainly on the different exchange behavior of the backbone amides in the two redox forms of the protein [3 -6,9-13].Heteronuclear NMR spin relaxation spectroscopy constitutes a powerful experimental approach for characterizing time-dependent conformational fluctuations in proteins and so represents a key to the understanding of different biophysical phenomena, including thermodynamic stability, folding, molecular recognition, and catalysis [14,15].However, the isotopically labeled mitochondrial cytochrome c required for these studies has been difficult to obtain due to the problem of expression of the native holoprotein in the preferred host, E. coli. 15N-Labeled samples of this protein have previously been obtained by two different strategies [13,16,17]. E. coli normally produces c-type cytochromes in the periplasm and recently heterologous expression has yielded 10 -15 mg : L 21 of such cytochromes [18]. In the present work to give improved yields we have used a nov...

Almost without exception, c-type cytochromes have heme covalently attached via two thioether linkages to the cysteine residues of a CXXCH motif. The reasons for the covalent attachment are not understood. Reported here is cytoplasmic expression in Escherichia coli of AXXCH and CXXAH variants of cytochrome c 552 from Hydrogenobacter thermophilus; remarkably, the single thioether bond proteins have, apart from an altered visible absorption spectrum, almost identical properties, including thermal stability and reduction potential, to the wild type CXXCH protein. In combination with previous work showing that an AXXAH variant of cytochrome c 552 is much less stable than the CXXCH form, it can be concluded that covalent attachment of heme via either of thioether bonds is sufficient to confer considerable stability and that these bonds contribute little to the setting of the reduction potential. The absence of AXXCH or CXXAH heme-binding motifs from bacterial cytochromes c may relate to the coexistence of the assembly pathway with that for formation of disulfide bonds in the bacterial periplasm.

Cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a Cys-XaaXaa-Cys-His peptide motif. In many Gram-negative bacteria, the heme is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c 552 , which has a normal CXXCH heme-binding motif, and variants with AXXCH, CXXAH, and AXXAH motifs, can be expressed as stable holocytochromes in the cytoplasm of Escherichia coli. By targeting these proteins to the periplasm using a signal peptide, with or without co-expression of the Ccm proteins, we have assessed the ability of the Ccm system to attach heme to proteins with no, one, or two cysteine residues in the heme-binding motif. Only the wild-type protein, with two cysteines, was effectively processed and thus accumulated in the periplasm as a holocytochrome. This is strong evidence for disulfide bond formation involving the two cysteine residues of apocytochrome c as an intermediate in Ccm-type Gramnegative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus.

This perspective seeks to discuss why biology often modifies the fundamental iron-protoporphyrin IX moiety that is the very versatile cofactor of many heme proteins. A very common modification is the attachment of this cofactor via covalent bonds to two (or rarely one) sulfur atoms of cysteine residue side chains. This modification results in c-type cytochromes, which have diverse structures and functions. The covalent bonds are made in different ways depending on the cell type. There is little understanding of the reasons for this complexity in assembly routes but proposals for the rationale behind the covalent modification are presented. In contrast to the widespread c-type cytochromes, the d1 heme is restricted to a single enzyme, the cytochrome cd1 nitrite reductase that catalyses the one-electron reduction of nitrite to nitric oxide. This is an extensively derivatised heme; a comparison is drawn with another type of respiratory nitrite reductase in which the active site is a c-type heme, but the product ammonia.

NMR techniques and 8-anilino-1-napthalenesulphonate (ANS) binding studies have been used to characterize the apo state of a variant of cytochrome c 552 from Hydrogenobacter thermophilus. In this variant the two cysteines that form covalent thioether linkages to the heme group have been replaced by alanine residues (C11A/C14A). CD studies show that the apo state contains ϳ14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R h , 16.6 Å). The apo state binds 1 mol of ANS/mol of protein, and a linear reduction in fluorescence enhancement is observed on adding aliquots of hemin to a solution of apo C11A/C14A cytochrome c 552 with ANS bound. These results suggest that the bound ANS is located in the heme binding pocket, which would therefore be at least partially formed in the apo state. Consistent with these characteristics, the formation of the holo state of the variant cytochrome c 552 from the apo state on the addition of heme has been demonstrated using NMR techniques.

Apocytochrome ~5 5 0 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c-type cytochromes, produces reduced levels of siderophores and carries a T n 5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondria1 haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates a t least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli y-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-def icient mutant, the apo form of P. denitrificans cytochrome css0 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.