Stereoselective in vitro formation of c-type cytochrome variants from Hydrogenobacter thermophilus containing only a single thioether bond

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.     

Yeast iso-1-cytochrome c: genetic analysis of structural requirements

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso-1-cytochrome c was facilitated by the recently resolved three dimensional structure of iso-1-cytochrome c [(1987) J. Mol. Biol. 199, 295-314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso-1-cytochrome c function appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso-1-cytochrome c.     

Spectroscopic properties of a mitochondrial cytochrome C with a single thioether bond to the heme prosthetic group

The yeast iso-1-cytochrome c variant Cys14Ser has been prepared in which one of the two thioether bonds by which the heme prosthetic group is normally bound to the protein has been eliminated. Comparison of the properties of this variant with those of the wild-type cytochrome provides insight into the role of this covalent attachment of the heme group to the apo-protein toward the functional properties of the wild-type cytochrome. Although NMR and EPR spectra indicate that the Cys14Ser variant ferricytochrome adopts the native conformation characteristic of the wild-type protein with His18 and Met80 coordinated to the heme iron (Met80 epsilon-CH -23.6 ppm; g(z), g(y), g(x), at 3.01, 2.29, approximately 1.3, respectively), the electronic spectrum of the variant does not exhibit the 695 nm CT band that is characteristic of native ferricytochromes c with these axial ligands. Chromatographic and spectropolarimetric comparison of the variant and wild-type ferricytochromes suggests that the structure of the variant is more disordered, particularly in the region of the sole tryptophanyl residue (Trp59). Upon reduction, the electronic spectrum of the variant exhibits a symmetrically broadened alpha-band that is shifted approximately 3 nm to the ultraviolet relative to its position in the spectrum in the wild-type protein. In the MCD spectrum, a band appears above 390 nm that is more intense than the Soret A-term which is also shifted to lower energy.     

A possible role for the covalent heme-protein linkage in cytochrome c revealed via comparison of N-acetylmicroperoxidase-8 and a synthetic, monohistidine-coordinated heme peptide

N-Acetylmicroperoxidase-8 (1) contains heme and residues 14-21 of horse mitochondrial cytochrome c (cyt c). The two thioether bonds linking protein to heme in cyt c are present in 1, and the native axial ligand His-18 remains coordinated to iron. As an approach to probing structural or functional roles played by the double covalent heme-protein linkage in cyt c, we have initiated a study in which the properties of 1 are compared with those of a synthetic mono-His coordinated heme peptide containing a single covalent linkage (2). One consequence of the greater conformational restriction imposed on peptide conformation in 1 is that His-Fe(III) coordination is approximately 1.4 kcal/mol more favorable in 1 than in 2. This highlights a clear advantage conferred to cyt c by having two covalent heme-protein linkages rather than one: greater thermodynamic stability of the protein fold. EPR (11 K) and resonance Raman (298 K) studies reveal that 1 and 2 exhibit a thermal high-spin/low-spin ferric equilibrium but that low-spin character is considerably more pronounced in 1. In addition, the thioether 2-(methylthio)ethanol (MTE) coordinates 0.5 kcal/mol more strongly to 1 than to 2 in 60:40 H(2)O/CH(3)OH and only triggers the expected conversion of iron to the low-spin state characteristic of ferric cyt c in the case of 1. This demonstrates that the axial ligand field provided by an imidazole and a thioether is too weak to induce a high-spin to low-spin conversion in a ferric porphyrin. Our results suggest that a conformationally constrained double covalent heme-protein linkage, as exists in 1 and its parent protein cyt c, is an effective solution that nature has evolved to circumvent this limitation. We propose that the stronger His-Fe(III) coordination enabled by such a linkage serves to markedly enhance the effective ligand field strength of His-18. Our studies with 1 and 2 suggest that a double covalent linkage in cyt c may also enable energetically more favorable trans ligation of Met-80 than would be possible if only a single linkage were present. This would serve to further increase the stability of the protein fold and perhaps to increase the effective ligand field strength of Met-80 as well.     

Characterization of the high-spin heme x in the cytochrome b6f complex of oxygenic photosynthesis

X-ray structures at 3.0-3.1 A resolution of the cytochrome b(6) f complex from the cyanobacterium Mastigocladus laminosus [Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Science 302, 1009-1014] and the green alga Chlamydomonas reinhardtii [Stroebel, D., Choquet, Y., Popot, J.-L., and Picot, D. (2003) Nature 426, 413-418] showed the presence of a unique heme, hemex, that is covalently linked by a single thioether bond to a Cys residue (Cys35) on the electrochemically negative (n) side of the cytochrome b(6) polypeptide. Heme x faces the intermonomer quinone exchange cavity. The only axial ligand associated with this heme is a H(2)O or OH(-) that is H-bonded to the propionate of the stromal side heme b(n), showing that it is pentacoordinate. The spectral properties of this heme were hardly defined at the time of the structure determination. The pyridine hemochromagen redox difference spectrum for heme x covalently bound to the cytochrome b polypeptide isolated from SDS-PAGE displays a low-amplitude broad spectrum with a peak at 553 nm, similar to that of other hemes with a single thioether linkage. The binding of CO and a hydrophobic cyanide analogue, butyl isocyanide, to dithionite-reduced b(6) f complex perturbs and significantly shifts the redox difference visible spectrum. Together with EPR spectra displaying g values of the oxidized complex of 6.7 and 7.4, heme x is defined as a ferric high-spin heme in a rhombic environment. In addition to a possible function in photosystem I-linked cyclic electron transport, the five-coordinate state implies that there is at least one more function of heme x that is related to axial binding of a physiological ligand.     

The cytochrome c fold can be attained from a compact apo state by occupancy of a nascent heme binding site

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 approximately 14% helical secondary structure, and measurements of hydrodynamic radii using pulse field gradient NMR methods show that it is compact (R(h), 16.6 A). 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. The properties of the apo state of C11A/C14A cytochrome c(552) reported here contrast strongly with those of mitochondrial cytochrome c whose apo state resembles a random coil under similar conditions.     

The Escherichia coli cytochrome c maturation (Ccm) system does not detectably attach heme to single cysteine variants of an apocytochrome c

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.     

Role of heme in structural organization of cytochrome c probed by semisynthesis

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.     

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.     

Characterization of Rhodobacter sphaeroides cytochrome c(2) proteins with altered heme attachment sites

In c-type cytochromes, heme is attached to the polypeptide via thioether linkages between vinyl groups on the tetrapyrrole ring and cysteine thiols in a CX(2)CH motif. To study the role of the heme-binding site in c-type cytochrome assembly and function, we generated amino acid changes in this region of Rhodobacter sphaeroides cytochrome c(2) ((15)Cys-Gln-Thr-Cys-His(19)). Amino acid substitutions at Cys(15), Cys(18), or His(19) produced mutant proteins that did not support growth via photosynthesis where this electron carrier is required. Many of these changes appeared to slow signal peptide removal, suggesting that heme attachment is coupled to processing of the c-type cytochrome precursor protein. Inserting an alanine between the cysteine ligands (CycA-Ins17A) did not significantly alter the behavior of this protein in vivo and in vitro, suggesting that the existence of 2 residues between cysteine thiols is not essential for heme attachment to a Class I c-type cytochrome like cytochrome c(2).     

Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

Biogenesis of cytochrome c1. Role of cytochrome c1 heme lyase and of the two proteolytic processing steps during import into mitochondria

The biogenesis of cytochrome c1 involves a number of steps including: synthesis as a precursor with a bipartite signal sequence, transfer across the outer and inner mitochondrial membranes, removal of the first part of the presequence in the matrix, reexport to the outer surface of the inner membrane, covalent addition of heme, and removal of the remainder of the presequence. In this report we have focused on the steps of heme addition, catalyzed by cytochrome c1 heme lyase, and of proteolytic processing during cytochrome c1 import into mitochondria. Following translocation from the matrix side to the intermembrane-space side of the inner membrane, apocytochrome c1 forms a complex with cytochrome c1 heme lyase, and then holocytochrome c1 formation occurs. Holocytochrome c1 formation can also be observed in detergent-solubilized preparations of mitochondria, but only after apocytochrome c1 has first interacted with cytochrome c1 heme lyase to produce this complex. Heme linkage takes place on the intermembrane-space side of the inner mitochondrial membrane and is dependent on NADH plus a cytosolic cofactor that can be replaced by flavin nucleotides. NADH and FMN appear to be necessary for reduction of heme prior to its linkage to apocytochrome c1. The second proteolytic processing of cytochrome c1 does not take place unless the covalent linkage of heme to apocytochrome c1 precedes it. On the other hand, the cytochrome c1 heme lyase reaction itself does not require that processing of the cytochrome c1 precursor to intermediate size cytochrome c1 takes place first. In conclusion, cytochrome c1 heme lyase catalyzes an essential step in the import pathway of cytochrome c1, but it is not involved in the transmembrane movement of the precursor polypeptide. This is in contrast to the case for cytochrome c in which heme addition is coupled to its transport directly across the outer membrane into the intermembrane space.     

Design and synthesis of de novo cytochromes c

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.     

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.     

In vitro studies on thioether bond formation between Hydrogenobacter thermophilus apocytochrome c(552) with metalloprotoporphyrin derivatives

Previously, in vitro formation of thioether bonds between Hydrogenobacter thermophilus apocytochrome c(552) and Fe-protoporphyrin IX has been demonstrated. Now we report studies on the reaction between the metalloderivatives Zn-, Co-, and Mn-protoporphyrin IX and the cysteine thiols of H. thermophilus apocytochrome c(552). All of these metalloporphyrins were capable of forming a "b-type cytochrome" state in which the hydrophobic prosthetic group is bound non-covalently. Zn(II)-protoporphyrin IX attached to the polypeptide covalently in the presence of either dithiothreitol or tri(2-carboxyethyl)phosphine to keep the thiol moieties reduced. These data show that the chemical nature of the thiol-reducing agent does not interfere with the thioether bond-forming mechanism. Mn-porphyrin could only react with the protein in the divalent state of the metal ion. Co-porphyrin did not react with the cysteine thiols of the apocytochrome in either oxidation state of the metal. In the absence of a metal (i.e. protoporphyrin IX itself), no reactivity toward apocytochrome is observed. These results have significant implications for the chemical requirements for thioether bond formation of heme vinyl groups to cysteine thiols and also have potential applications in de novo design of metalloproteins.     

Cytochrome c maturation system on the negative side of bioenergetic membranes: CCB or System IV

Cytochromes of the c-type contain hemes covalently attached via one or, more generally, two thioether bonds between the vinyls of heme b and the thiols of cysteine residues of apocytochromes. This post-translational modification relies on membrane-associated specific biogenesis proteins, referred to as cytochrome c maturation systems. At least three different versions (i.e. Systems I-III) are found on the positive side of bioenergetic membranes in different organisms and compartments. The present minireview is concerned with systems on the negative side of the membranes. It describes System IV, also referred to as cofactor assembly on complex C subunit B, for heme binding on cytochrome b(6) through one thioether bond; this covalent heme is usually called c(i) . This system is found in all organisms with oxygenic photosynthesis but not in Firmicutes, although they also have a cytochrome b protein with an additional heme c(i) covalently attached via a single thioether bond.     

Loss of either of the two heme-binding cysteines from a class I c-type cytochrome has a surprisingly small effect on physicochemical properties

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.     

Molecular modeling of cytochrome b₅ with a single cytochrome c-like thioether linkage

Bovine liver cytochrome b ₅ (cyt b ₅), with heme bound noncovalently, has been converted into a cyt c-like protein (cyt b ₅ N57C) by constructing a thioether linkage between the heme and the engineered cysteine residue. With no X-ray or NMR structure available, we herein performed a molecular modeling study of cyt b ₅ N57C. On the other hand, using amino acid sequence information for a newly discovered member of the cyt b ₅ family, domestic silkworm cyt b ₅ (DS cyt b ₅), we predicted the protein structure by homology modeling in combination with MD simulation. The modeling structure shows that both Cys57 in cyt b ₅ N57C, and Cys56, a naturally occurring cysteine in DS cyt b ₅, have suitable orientations to form a thioether bond with the heme 4-vinyl group, as the heme is in orientation A. In addition to providing structural information that was not previously obtained experimentally, these modeling studies provide insight into the formation of cyt c-like thioether linkages in cytochromes, and suggest that c-type cyt b ₅ maturation involves a b-type intermediate.     

Regions of Rhodobacter sphaeroides cytochrome c2 required for export, heme attachment, and function.

Cytochrome c2 is a periplasmic redox protein involved in both the aerobic and photosynthetic electron transport chains of Rhodobacter sphaeroides. The process of cytochrome c2 maturation has been analyzed in order to understand the protein sequences involved in attachment of the essential heme moiety to the cytochrome c2 polypeptide and localization of the protein to the periplasm. To accomplish this, five different translational fusions which differ only in the cytochrome c2 fusion junction were constructed between cytochrome c2 and the Escherichia coli periplasmic alkaline phosphatase. All five of the fusion proteins are exported to the periplasmic space. The four fusion proteins that contain the NH2-terminal site of covalent heme attachment to cytochrome c2 are substrates for heme binding, suggesting that the COOH-terminal region of the protein is not required for heme attachment. Three of these hybrids possess heme peroxidase activity, which indicates that they are functional as electron carriers. Biological activity is possessed by one hybrid protein constructed five amino acids before the cytochrome c2 COOH terminus, since synthesis of this protein restores photosynthetic growth to a photosynthetically incompetent cytochrome c2-deficient derivative of R. sphaeroides. Biochemical analysis of these hybrids has confirmed CycA polypeptide sequences sufficient for export of the protein (A. R. Varga and S. Kaplan, J. Bacteriol. 171:5830-5839, 1989), and it has allowed us to identify regions of the protein sufficient for covalent heme attachment, heme peroxidase activity, docking to membrane-bound redox partners, or the capability to function as an electron carrier.     

Characterization of recombinant horse cytochrome c synthesized with the assistance of Escherichia coli cytochrome c maturation factors

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.