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.     

Cyc2p, a membrane-bound flavoprotein involved in the maturation of mitochondrial c-type cytochromes

Mitochondrial apocytochrome c and c1 are converted to their holoforms in the intermembrane space by attachment of heme to the cysteines of the CXXCH motif through the activity of assembly factors cytochrome c heme lyase and cytochrome c1 heme lyase (CCHL and CC1HL). The maintenance of apocytochrome sulfhydryls and heme substrates in a reduced state is critical for the ligation of heme. Factors that control the redox chemistry of the heme attachment reaction to apocytochrome c are known in bacteria and plastids but not in mitochondria. We have explored the function of Cyc2p, a candidate redox cytochrome c assembly component in yeast mitochondria. We show that Cyc2p is required for the activity of CCHL toward apocytochrome c and c1 and becomes essential for the heme attachment to apocytochrome c1 carrying a CAPCH instead of CAACH heme binding site. A redox function for Cyc2p in the heme lyase reaction is suggested from 1) the presence of a noncovalently bound FAD molecule in the C-terminal domain of Cyc2p, 2) the localization of Cyc2p in the inner membrane with the FAD binding domain exposed to the intermembrane space, and 3) the ability of recombinant Cyc2p to carry the NADPH-dependent reduction of ferricyanide. We postulate that, in vivo, Cyc2p interacts with CCHL and is involved in the reduction of heme prior to its ligation to apocytochrome c by CCHL.     

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.     

The biosynthesis of bacterial and plastidic c-type cytochromes

The biosynthesis of bacterial and plastidic c-type cytochromes includes several steps that occur post-translationally. In the case of bacterial cytochromes, the cytosolically synthesized pre-proteins are translocated across the cytoplasmic membrane, the pre-proteins are cleaved to their mature forms and heme is ligated to the processed apoprotein. Although heme attachment has not been studied extensively at the biochemical level, molecular genetic approaches suggest that the reaction generally occurs after translocation of the apoprotein to the periplasm. Recent studies with Bradyrhizobium japonicum and Rhodobacter capsulatus indicate that the process of heme attachment requires the function of a large number of genes. Mutation of these genes generates a pleiotropic deficiency in all c-type cytochromes, suggesting that the gene products participate in processes required for the biosynthesis of all c-type cytochromes. In eukaryotic cells, the biosynthesis of photosynthetic c-type cytochromes is somewhat more complex owing to the additional level of compartmentation. Nevertheless, the basic features of the pathway appear to be conserved. For instance, as is the case in bacteria, translocation and processing of the pre-proteins is not dependent on heme attachment. Genetic analysis suggests that the nuclear as well as the plastid genomes encode functions required for heme attachment, and that these genes function in the biosynthesis of the membrane-associated as well as the soluble c-type cytochrome of chloroplasts. A feature of cytochromes c biogenesis that appears to be conserved between chloroplasts and mitochondria is the sub-cellular location of the heme attachment reaction (p-side of the energy transducing membrane). Continued investigation of all three experimental systems (bacteria, chloroplasts, mitochondria) is likely to lead to a greater understanding of the biochemistry of cytochrome maturation as well as the more general problem of cofactor-protein association during the assembly of an energy transducing membrane.Abbreviations CCHL cytochrome c/heme lyase - CC₁HL cytochrome cl/heme lyase - cyt cytochrome - EMS ethyl methane sulphonate - n-side electrochemically negative side of an energy transducing membrane - p-side electrochemically positive side of an energy transducing membrane - PhoA alkaline phosphatase (encoded by the phoA locus)     

Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes

Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA:phoA and ccdA :lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R. capsulatus.     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M_r molecular weight - pI isoelectric point     

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.     

A systematic investigation of multiheme c-type cytochromes in prokaryotes

Multiheme c-type cytochromes (MHCs) are metalloproteins that can play various biochemical roles, including enzymatic activity and electron transfer. As electron transfer proteins, the presence of multiple heme cofactors in the vicinity allows electrons to rapidly travel relatively long distances. MHCs are often characterized by relatively low structural complexity, with the heme cofactors being largely responsible for maintaining the structure in place, owing to the protein–heme covalent linkages. In this work, we analyzed an extensive ensemble of 594 complete prokaryotic proteomes, amounting to more than 1.9 million sequences, to characterize their content in MHCs. We identified 1,659 MHCs in 258 organisms. The presence of MHCs was found to correlate quite well with the capability of an organism to synthesize or take up heme. For two organisms, the presence of MHCs in the proteome could be taken as a hint to the presence of divergent heme uptake pathways. The most common numbers of heme-binding motifs in a sequence were four (25%) and two (23%), followed by five (13%) and ten (9.8%). The average protein-to-heme ratio was relatively similar for all MHCs, except diheme proteins, regardless of the number of motifs at around 60 ± 30. The latter ratio could in favorable cases be a useful indicator for functional assignments of novel MHCs. Finally, we showed that the amount of structural information currently available for MHCs is limited with respect to the diversity of this broad class of metalloproteins. Experimental efforts in the structural investigation of MHCs are thus warranted.     

Fluorescent gels as a general technique for characterizing bacterial c-type cytochromes

The technique described by Katan (Anal. Biochem. 74, 1976, 132–137) for detecting c-type cytochromes on dodecyl sulfate/polyacrylamide gels by their red fluorescence has been adapted for use with bacterial extracts. No purification is required, except for organic solvent treatment to remove lipids. A wide range of c-type cytochromes has been found to give very similar red fluorescence. Molecular weights can be estimated by means of dansylated marker proteins fluorescing green.     

Electrostatic effects on the kinetics of oxidation-reduction reactions of c-type cytochromes.

The kinetics of the oxidation-reduction reactions between horse heart cytochrome c, Euglena gracilis cytochrome c552, and ions (ascorbate, ferricyanide, and ferrocyanide) was investigated as a function of ionic strength at pH 7, 25 degrees C. The ionic strength was varied between 0.002 and 0.02 M. Data were analyzed according to four different functions of ionic strength. Results showed that the Kirkwood-Tanford smeared charge model holds well for the calculation of the activity coefficients and that the whole charges of these proteins are reflected in the rates of their reactions. Chemical modifications or changes in the pH that altered the charge of the proteins affected the primary salt effects as predicted by the smeared charge model.     

Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: Purification and properties

Abstract Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans . It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, E _m,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c , cytochrome c -550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c -552 (m) and c -55 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c -550 (m) was higher than that of cytochrome c -552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c -552 (m) was greatly lower than that of cytochrome c -552 (s).     

Measurement of the oxidation-reduction potentials of amicyanin and c-type cytochromes from Paracoccus denitrificans

The oxidation-reduction potentials of four periplasmic electron carrier proteins from Paracoccus denitrificans have been determined. Their midpoint potentials are: amicyanin, 294 +/- 6 mV; cytochrome c-550, 253 +/- 5 mV; cytochrome c-551i, 190 +/- 4 mV; and cytochrome c-553i, 148 +/- 5 mV. Although rapid amicyanin-mediated transfer of electrons from methylamine dehydrogenase to cytochrome c-551i was observed, reduced amicyanin did not reduce oxidized cytochrome c-551i in the absence of methylamine dehydrogenase.     

Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry

We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.     

Resonance Raman study of multihemic c-type cytochromes from Desulfuromonas acetoxidans.

Two multihemic cytochromes c from the sulfur reducing bacteria Desulfuromonas acetoxidans have been studied by optical and resonance Raman spectroscopy: cytochrome c551.5, a trihemic cytochrome and cytochrome c Mr 50 000, a recently isolated high molecular mass cytochrome. The redox and Raman characteristics of cytochrome c551.5 are compared to those of the tetrahemic cytochromes c3 from Desulfovibrio. While the redox behavior, followed by spectroelectrochemistry, is similar to that of cytochrome c3, showing the same conformational change after reduction of the highest potential heme, the Raman data show a contribution from a His- form of the axial ligands and lead to the assignment of a band at 218 cm-1 to the Fe(III)-(His)2 stretching vibration. The Raman data on cytochrome c Mr 50 000 are in favor of an entirely low spin species with two different sets of axial ligands. A partially reduced state is easily accessible by ascorbate addition.