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1.
In vitro formation of Hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). Now we report that the single cysteine variants of H. thermophilus c552, which bind heme via a single thioether bond, also form in vitro. Furthermore, reaction of the apocytochromes containing either AXXCH or CXXAH in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroheme resulted predominantly in covalent attachment between Cys-11 and the 2-vinyl moiety and Cys-14 and the 4-vinyl functionality. This observation shows that the covalent attachment of heme to apocytochrome is stereoselective, indicating that the initial non-covalent complexes between apoprotein and heme have to be in the correct stereochemical orientation for preferential promotion of thioether bond formation. Additionally, the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme were reacted with wild-type H. thermophilus c552 to yield another modification of cytochromes containing only one thioether bond. These results show that the formation of the two thioether bonds in typical c-type cytochromes can occur independently from one another. Aspects of rotational isomerism of heme in heme-proteins are discussed.  相似文献   

2.
Cytochromes c are typically characterized by the covalent attachment of heme to polypeptide through two thioether bonds with the cysteine residues of a Cys-Xaa-Xaa-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 Gram-negative bacterial cytochrome c biogenesis and/or that only a pair of cysteines can be recognized by the heme attachment apparatus.  相似文献   

3.
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.  相似文献   

4.
Cytochromes c are metalloproteins that function in electron transfer reactions and contain a heme moiety covalently attached via thioether linkages between the co-factor and a CXXCH motif in the protein. Covalent attachment of the heme group occurs on the positive side of all energy-transducing membranes (bacterial periplasm, mitochondrial intermembrane space and thylakoid lumen) and requires minimally: 1) synthesis and translocation of the apocytochromes c and heme across at least one biological membrane, 2) reduction of apocytochromes c and heme and maintenance under a reduced form prior to 3) catalysis of the heme attachment reaction. Surprisingly, the conversion of apoforms of cytochromes c to their respective holoforms occurs through at least three different pathways (systems I, II and III). In this review, we detail the assembly process of soluble cytochrome c and membrane-bound cytochrome c1, the only two mitochondrial c-type cytochromes that function in respiration. Mitochondrial c-type cytochromes are matured in the intermembrane space via the system I or system III pathway, an intriguing finding considering that the biochemical requirements for cytochrome c maturation are believed to be common regardless of the energy-transducing membrane under study.  相似文献   

5.
The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome. Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif. In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly. We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts. The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes. Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues. Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b(6)f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen. We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway.  相似文献   

6.
Hemes c are characterized by their covalent attachment to a polypeptide via a widely conserved CXXCH motif. There are multiple biological systems that facilitate heme c biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromes c in Escherichia coli-based expression systems. System III, cytochrome c heme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophilus cytochrome c(552) variants was successfully expressed and matured by the CCM system with CX(n)CH motifs where n = 1-4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1-3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemes c with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL.  相似文献   

7.
Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.  相似文献   

8.
Summary: Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe+3 state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe+2) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.  相似文献   

9.
Cytochromes c are characterized by the presence of a protoporphyrin IX group covalently attached to the polypeptide via one or two thioether bonds to Cys side chains. The heme attachment process, known as cytochrome c maturation, occurs posttranslationally in the periplasm (for bacterial cytochromes c) or in the mitochondrial intermembrane space (for eukaryotic cytochromes c) through a pathway dependent on the organism. It is demonstrated in this work that a mitochondrial cytochrome c expressed in Escherichia coli that undergoes maturation under control of the E. coli cytochrome c maturation factors achieves a native-like structure and stability. The recombinant protein is characterized spectroscopically (by circular dichroism (CD), absorption, and nuclear magnetic resonance (NMR) spectroscopy) and it is verified that the heme and its environment are indistinguishable from authentic horse cytochrome c. Mass spectrometry reveals that the recombinant protein is not acetylated at the N terminus, however, no significant effect on protein structure or stability is detected as a result.  相似文献   

10.
Simon J  Hederstedt L 《The FEBS journal》2011,278(22):4179-4188
Organisms employ one of several different enzyme systems to mature cytochromes c. The biosynthetic process involves the periplasmic reduction of cysteine residues in the heme c attachment motif of the apocytochrome, transmembrane transport of heme b and stereospecific covalent heme attachment via thioether bonds. The biogenesis System II (or Ccs system) is employed by β-, δ- and ε-proteobacteria, Gram-positive bacteria, Aquificales and cyanobacteria, as well as by algal and plant chloroplasts. System II comprises four (sometimes only three) membrane-bound proteins: CcsA (or ResC) and CcsB (ResB) are the components of the cytochrome c synthase, whereas CcdA and CcsX (ResA) function in the generation of a reduced heme c attachment motif. Some ε-proteobacteria contain CcsBA fusion proteins constituting single polypeptide cytochrome c synthases especially amenable for functional studies. This minireview highlights the recent findings on the structure, function and specificity of individual System II components and outlines the future challenges that remain to our understanding of the fascinating post-translational protein maturation process in more detail.  相似文献   

11.
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.  相似文献   

12.
C-type cytochromes are a structurally diverse group of haemoproteins, which are related by the occurrence of haem covalently attached to a polypeptide via two thioether bonds formed by the vinyl groups of haem and cysteine side chains in a CXXCH peptide motif. Remarkably, three different post-translational systems for forming these cytochromes have been identified. The evolution of both the proteins themselves and the biogenesis systems poses many questions to which answers are currently being sought. In this article we review the progress that has been made in understanding the need for covalent attachment of haem to proteins in cytochromes c and the complex systems involved in their formation.  相似文献   

13.
Ishida M  Dohmae N  Shiro Y  Oku T  Iizuka T  Isogai Y 《Biochemistry》2004,43(30):9823-9833
Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.  相似文献   

14.
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).  相似文献   

15.
Hydrogen exchange (HX) rates and midpoint potentials (Em) of variants of cytochrome c from Pseudomonas aeruginosa (Pa cyt c551) and Hydrogenobacter thermophilus (Ht cyt c552) have been characterized in an effort to develop an understanding of the impact of properties of the Cys-X-X-Cys-His pentapeptide c-heme attachment (CXXCH) motif on heme redox potential. Despite structural conservation of the CXXCH motif, Ht cyt c552 exhibits a low level of protection from HX for amide protons within this motif relative to Pa cyt c551. Site-directed mutants have been prepared to determine the structural basis for and functional implications of these variations on HX behavior. The double mutant Ht-M13V/K22M displays suppressed HX within the CXXCH motif as well as a decreased Em (by 81 mV), whereas the corresponding double mutant of Pa cyt c551 (V13M/M22K) exhibits enhanced HX within the CXXCH pentapeptide and a modest increase in Em (by 30 mV). The changes in Em correlate with changes in axial His chemical shifts in the ferric proteins reflecting the extent of histidinate character. Thus, the mobility of the CXXCH pentapeptide is found to impact the His-Fe(III) interaction and therefore the heme redox potential.  相似文献   

16.
In bacterial c-type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c-binding motif. Here, a novel octa-haem c protein, MccA, is described that contains only seven conventional haem c-binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH signature. Mass spectrometric analysis of purified MccA from Wolinella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX15CH sequence involved in haem c binding. Spectroscopic characterization of MccA identified an unusual high-potential haem c with a red-shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the maturation of MccA in W. succinogenes. Equivalent haem lyase-encoding genes belonging to either the bacterial cytochrome c biogenesis system I or II are present in the vicinity of every known mccA gene suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer-based prediction of putative haem c-binding motifs in bacterial proteomes.  相似文献   

17.
Cytochromes c are widespread respiratory proteins characterized by the covalent attachment of heme. The formation of c-type cytochromes requires, in all but a few exceptional cases, the formation of two thioether bonds between the two cysteine sulfurs in a -CXXCH- motif in the protein and the vinyl groups of heme. The vinyl groups of the heme are not particularly activated and therefore the addition reaction does not physiologically occur spontaneously in cells. There are several diverse post-translational modification systems for forming these bonds. Here, we describe the complex multiprotein cytochrome c maturation (Ccm) system (in Escherichia coli comprising the proteins CcmABCDEFGH), also called System I, that performs the heme attachment. System I is found in plant mitochondria, archaea and many Gram-negative bacteria; the systems found in other organisms and organelles are described elsewhere in this minireview series.  相似文献   

18.
Kang X  Carey J 《Biochemistry》1999,38(48):15944-15951
The heme prosthetic group of cytochrome c is covalently attached to the protein through thioether bonds to two cysteine side chains. The role of covalent heme attachment to cytochrome c is not understood, and most heme proteins bind the prosthetic group by iron ion ligation and tertiary interactions only. A two-armed attachment seems redundant if the role of covalent connection is to limit heme group orientation or to decouple heme affinity from redox potential. These considerations suggested that one role for covalent attachment of the rigid planar heme might be in organizing the cytochrome c protein structure. Indeed, porphyrin cytochrome c (in which the heme iron ion has been removed) is substantially more ordered than apocytochrome c, having characteristics consistent with a molten globule state. To assess the importance of planar rigidity in ordering this protein, semisynthesis was used to substitute porphyrin by two hydrophobic surrogates, one based on biphenyl and the other on phenanthrene, which have different degrees of planarity and rigidity. The expected two-armed covalent attachment of each surrogate was confirmed in the protein products by a variety of methods including mass spectrometry and NMR. Despite being only about half the size of the porphyrin macrocycle, and lacking any possibility for ligation or polar group interactions with the surrounding protein, the two surrogates confer helix contents that are comparable to that of the molten globule formed by porphyrin cytochrome c under similar solution conditions. The pH titrations of the derivatives monitored by circular dichroism exhibit reversible, bell-shaped folding and unfolding transitions, implying that charge group interactions in the protein are involved in stabilizing the helical structures formed. The thermal transitions of the two derivatives at neutral pH are cooperative, with similar midpoints. The similarity of helical content and structural stability in the two derivatives indicates that the increase in conformational freedom by the biphenyl surrogate does not substantially reduce protein structural stability. The similarity of the two derivatives to porphyrin cytochrome c suggests that the common feature among the three covalently attached groups-their hydrophobicity-is by far the dominant factor in organizing stable structures in the protein.  相似文献   

19.
The covalent attachment of heme to mitochondrial cytochrome c is catalysed by holocytochrome c synthase (HCCS, also called heme lyase). How HCCS functions and recognises the substrate apocytochrome is unknown. Here we have examined HCCS recognition of a chimeric substrate comprising a short mitochondrial cytochrome c N-terminal region with the C-terminal sequence, including the CXXCH heme-binding motif, of a bacterial cytochrome c that is not otherwise processed by HCCS. Heme attachment to the chimera demonstrates the importance of the N-terminal region of the cytochrome. A series of variants of a mitochondrial cytochrome c with amino acid replacements in the N-terminal region have narrowed down the specificity determinants, providing insight into HCCS substrate recognition.  相似文献   

20.
Structural homology of cytochromes c.   总被引:1,自引:0,他引:1  
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.  相似文献   

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