Structural homology of cytochromes c.

Cytochromes c from many eukaryotic and diverse prokaryotic organisms have been investigated and compared using high-resolution nuclear magnetic resonance spectroscopy. Resonances have been assigned to a large number of specific groups, mostly in the immediate environment of the heme. This information, together with sequence data, has allowed a comparison of the heme environment and protein conformation for these cytochromes. All mitochondrial cytochromes c are found to be very similar to the cytochromes c2 from Rhodospirillaceae. In the smaller bacterial cytochromes, Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome c552, the orientation of groups near the heme is very similar, but the folding of the polypeptide chain is different. The heme environment of these two proteins is similar to that of the larger bacterial and mitochondrial cytochromes. Two low-potential cytochromes, Desulfovibrio vulgaris cytochrome c553 and cytochrome c554 from a halotolerant micrococcus have heme environments which are not very similar to those of the other proteins reported here.     

Cytochrome c maturation. The in vitro reactions of horse heart apocytochrome c and Paracoccus dentrificans apocytochrome c550 with heme

C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c(552) (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates. Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand. Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes. In vitro reaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.     

Coordination of the heme iron in the low-potential cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Different chirality of the axially bound methionine in the oxidized and reduced states

The coordination geometry at the heme iron of the cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans was investigated by 1H-nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with other c-type cytochromes. In contrast, a new structure was observed for the axial methionine in the reduced cytochromes c-553. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa and cytochrome c5 from Pseudomonas mendocina, which also contain S-chiral methionine, a different spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine prevails. For the ferricytochromes c-553 R chirality was found for the iron-bound sulfur. This is the first observation of different methionine chirality in different oxidation states of the same c-type cytochrome.     

Dynamic ligation properties of the Escherichia coli heme chaperone CcmE to non-covalently bound heme

The cytochrome c maturation protein CcmE is an essential membrane-anchored heme chaperone involved in the post-translational covalent attachment of heme to c-type cytochromes in Gram-negative bacteria such as Escherichia coli. Previous in vitro studies have shown that CcmE can bind heme both covalently (via a histidine residue) and non-covalently. In this work we present results on the latter form of heme binding to a soluble form of CcmE. Examination of a number of site-directed mutants of E. coli CcmE by resonance Raman spectroscopy has identified ligands of the heme iron and provided insight into the initial steps of heme binding by CcmE before it binds the heme covalently. The heme binding histidine (His-130) appears to ligate the heme iron in the ferric oxidation state, but two other residues ligate the iron in the ferrous form, thereby freeing His-130 to undergo covalent attachment to a heme vinyl group. It appears that the heme ligation in the non-covalent form is different from that in the holo-form, suggesting that a change in ligation could act as a trigger for the formation of the covalent bond and showing the dynamic and oxidation state-sensitive ligation properties of CcmE.     

Proteins involved in maturation pathways of plant mitochondrial and plastid c-type cytochromes

c-type cytochromes are characterized by the presence of two covalent bonds linking heme to apocytochrome and by the heme attachment motif in the apoprotein. Several molecular systems for the maturation of c-type cytochromes have evolved in different organisms. The best characterized are three of them: system I, system II and system III. Heme is synthesized in bacterial cytoplasm, in plastids, and in animal and fungal mitochondria. Therefore the maturation of bacterial and plastid c-type cytochromes involves the transport of heme and apocytochrome from the n-side to the p-side of the respective biological membranes and the formation of the covalent bond at the p-side. It should be underlined that the site of the c-type apocytochrome synthesis is also distinct from the site of its functioning. The aim of this review is to present the current state of knowledge concerning the structure and function of two systems - system I and system II - in the maturation of plant mitochondrial and plastid c-type cytochromes, respectively.     

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.     

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.     

Structural and functional approach toward a classification of the complex cytochrome c system found in sulfate-reducing bacteria.

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.     

A new spatial structure for the axial methionine observed in cytochrome c5 from Pseudomonas mendocina. Correlations with the electronic structure of heme c

Cytochrome c5 from Pseudomonas mendocina has been isolated and the coordination geometry at the heme iron was investigated by 1H nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with that of other c-type cytochromes. In contrast, a new structure was observed for the axial methionine. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa, which also contain S-chiral methionine, the spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine is markedly different. Analysis of the electron spin density distribution in ferricytochrome c5 in the light of this new coordination geometry provides additional support for the hypothesis that the electronic structure of heme c is primarily governed by the orientation of the sp3 lone-pair orbital of the axial sulfur atom with respect to the heme plane.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

A screen for genes of heme uptake identifies the FLC family required for import of FAD into the endoplasmic reticulum

Although Candida albicans and Saccharomyces cerevisiae express very similar systems of iron uptake, these species differ in their capacity to use heme as a nutritional iron source. Whereas C. albicans efficiently takes up heme, S. cerevisiae grows poorly on media containing heme as the sole source of iron. We identified a gene from C. albicans that would enhance heme uptake when expressed in S. cerevisiae. Overexpression of CaFLC1 (for flavin carrier 1) stimulated the growth of S. cerevisiae on media containing heme iron. In C. albicans, deletion of both alleles of CaFLC1 resulted in a decrease in heme uptake activity, whereas overexpression of CaFLC1 resulted in an increase in heme uptake. The S. cerevisiae genome contains three genes with homology to CaFLC1, and two of these, termed FLC1 and FLC2, also stimulated growth on heme when overexpressed in S. cerevisiae. The S. cerevisiae Flc proteins were detected in the endoplasmic reticulum and the FLC genes encoded an essential function, as strains deleted for either FLC1 or FLC2 were viable, but deletion of both FLC1 and FLC2 was synthetically lethal. FLC gene deletion resulted in pleiotropic phenotypes related to defects in cell wall integrity. High copy suppressors of this synthetic lethality included three mannosyltransferases, VAN1, KTR4, and HOC1. FLC deletion strains exhibited loss of cell wall mannose phosphates, defects in cell wall assembly, and delayed maturation of carboxypeptidase Y. Permeabilized cells lacking FLC proteins exhibited dramatic loss of FAD import activity. We propose that the FLC genes are required for import of FAD into the lumen of the endoplasmic reticulum, where it is required for disulfide bond formation.     

Cyanide reactivity of cytochrome c derivatives.

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivative demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10-20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 . 10(-2) s-1.     

Individual 1H-NMR assignments for the heme groups and the axially bound amino acids and determination of the coordination geometry at the heme iron in a mixture of two isocytochromes c-551 from Rhodopseudomonas gelatinosa

This paper describes chemical and physicochemical studies of two small isocytochromes c-551 (approx. 9000 dalton) from Rhodopseudomonas gelatinosa. In spite of numerous amino acid substitutions in the N-terminal half of the sequence the two isoproteins could not be separated by the procedures used, presumably because they have identical size, charge and isoelectric points. Individual assignments of the 1H-NMR lines of heme c and the axial ligands to the heme iron were therefore obtained by nuclear Overhauser enhancement measurements and saturation transfer experiments in a mixed solution of the two isocytochromes c-551. The conformation of the coordination sphere was investigated by additional 1H-NMR and circular dichroism studies. For both isoproteins the electronic structure of the heme and the chirality of the methionine attachment to the iron were found to coincide with those in Pseudomonas cytochromes c-551, i.e., S chirality was observed for the axial methionine. The Rps. gelatinosa cytochromes c-551 thus differ from mammalian, yeast, Euglena gracilis and Rhodospirillum rubrum cytochromes c, which all have R chirality at the axial methionine and concomitantly a characteristically different electronic heme structure. This is the first observation of S chirality of the axially bound methionine in a species outside the Pseudomonas family. The redox potentials of the two isocytochromes c-551 of Rps. gelatinosa differ by approx. 120 mV, and there is no cross-exchange of electrons between the two species. The two isoproteins could thus function in two different, parallel electron-transfer chains or at two different locations in a single transfer sequence.     

Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens

Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.     

Resolving the individual components of a pH-induced conformational change

This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves. We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c. The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations. By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond. We also noted that, in the horse and untrimethylated S. cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves. The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.     

Order within a mosaic distribution of mitochondrial c-type cytochrome biogenesis systems?

Mitochondrial cytochromes c and c(1) are present in all eukaryotes that use oxygen as the terminal electron acceptor in the respiratory chain. Maturation of c-type cytochromes requires covalent attachment of the heme cofactor to the protein, and there are at least five distinct biogenesis systems that catalyze this post-translational modification in different organisms and organelles. In this study, we use biochemical data, comparative genomic and structural bioinformatics investigations to provide a holistic view of mitochondrial c-type cytochrome biogenesis and its evolution. There are three pathways for mitochondrial c-type cytochrome maturation, only one of which is present in prokaryotes. We analyze the evolutionary distribution of these biogenesis systems, which include the Ccm system (System I) and the enzyme heme lyase (System III). We conclude that heme lyase evolved once and, in many lineages, replaced the multicomponent Ccm system (present in the proto-mitochondrial endosymbiont), probably as a consequence of lateral gene transfer. We find no evidence of a System III precursor in prokaryotes, and argue that System III is incompatible with multi-heme cytochromes common to bacteria, but absent from eukaryotes. The evolution of the eukaryotic-specific protein heme lyase is strikingly unusual, given that this protein provides a function (thioether bond formation) that is also ubiquitous in prokaryotes. The absence of any known c-type cytochrome biogenesis system from the sequenced genomes of various trypanosome species indicates the presence of a third distinct mitochondrial pathway. Interestingly, this system attaches heme to mitochondrial cytochromes c that contain only one cysteine residue, rather than the usual two, within the heme-binding motif. The isolation of single-cysteine-containing mitochondrial cytochromes c from free-living kinetoplastids, Euglena and the marine flagellate Diplonema papillatum suggests that this unique form of heme attachment is restricted to, but conserved throughout, the protist phylum Euglenozoa.     

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.     

Characterization of Hydrogenobacter thermophilus cytochromes c 552 expressed in the cytoplasm and periplasm of Escherichia coli

Hydrogenobacter thermophilus cytochrome c(552) ( Ht cyt c(552)) is a small monoheme protein in the cytochrome c(551) family. Ht cyt c(552) is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c(552) in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The (1)H NMR chemical shifts of the heme methyls of the oxidized ( S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c(552).     

Structures of cytochrome c-549 and cytochrome c6 from the cyanobacterium Arthrospira maxima

Cytochrome c(6) and cytochrome c-549 are small (89 and 130 amino acids, respectively) monoheme cytochromes that function in photosynthesis. They appear to have descended relatively recently from the same ancestral gene but have diverged to carry out very different functional roles, underscored by the large difference between their midpoint potentials of nearly 600 mV. We have determined the X-ray crystal structures of both proteins isolated from the cyanobacterium Arthrospira maxima. The two structures are remarkably similar, superimposing on backbone atoms with an rmsd of 0.7 A. Comparison of the two structures suggests that differences in solvent exposure of the heme and the electrostatic environment of the heme propionates, as well as in heme iron ligation, are the main determinants of midpoint potential in the two proteins. In addition, the crystal packing of both A. maxima cytochrome c-549 and cytochrome c(6) suggests that the proteins oligomerize. Finally, the cytochrome c-549 dimer we observe can be readily fit into the recently described model of cyanobacterial photosystem II.     

Functional characterization of the C-terminal domain of the cytochrome c maturation protein CcmE

CcmE is a heme chaperone involved in the periplasmic maturation of c-type cytochromes in many bacteria and plant mitochondria. It binds heme covalently and subsequently transfers it to the apo form of cytochromes c. To examine the role of the C-terminal domain of CcmE in the binding of heme, in vitro heme binding to the apo form of a truncated (immediately before Pro-136) version of the periplasmic domain of the heme chaperone from Escherichia coli was studied. Removal of the C-terminal domain dramatically altered the ligation of non-covalently bound heme in CcmE' (the soluble form lacking the membrane anchor) but only slightly affected its affinity for protoporphyrin IX and 8-anilino-1-naphthalenesulfonate. This finding has significant mechanistic implications for in vivo holo-CcmE formation and indicates that the C-terminal region is not required for the recruitment and docking of heme into its binding site but is likely to contain amino acid(s) involved in heme iron axial coordination. Removal of the C-domain significantly impaired in vivo heme binding to CcmE and conversion of apocytochrome to holoprotein by a similar factor, suggesting that the C-terminal domain of the chaperone is primarily involved in heme binding to CcmE rather than in heme transfer to the apo cytochrome.