Biogenesis of Respiratory Cytochromes in Bacteria (original) (raw)
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The biosynthesis of bacterial and plastidic c-type cytochromes
Photosynthesis Research, 1994
The biosynthesis of bacterial and plastidic c-type cytochromes includes several steps that occur post-translationaUy. 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 Bradyrhizobiumjaponicum 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 heine 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.
Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase
The EMBO Journal, 1989
Communicated by M.Wikstrom COIH is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COmI gene from Paracoccus denitificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blueshifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COIH complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIH gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COm and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intennediates of the assembly pathway or dissociation products of a labile COI + COIl complex and its conformational variants.
The Unfolding of Oxidized c-Type Cytochromes: The Instructive Case of Bacillus pasteurii
Journal of Molecular Biology, 2002
The reversible unfolding of oxidized Bacillus pasteurii cytochrome c 553 by guanidinium chloride under equilibrium conditions has been monitored by NMR and optical spectroscopy. The results obtained indicate that unfolding takes place through a mechanism involving the detachment from heme iron coordination of the sulfur of the Met71 axial ligand and yielding either a high spin (HS) or a low spin (LS 1) species, depending on the pH value. In the LS 1 form the Met71 is replaced by another protein ligand, possibly Lys. The ligand exchange reaction does not reach completion until the protein backbone reaches a largely unfolded state, as monitored through 1 H-15 N NMR experiments, thus demonstrating that there is a significant correlation between formation of the Fe-S bond and native structure stability. 1 H/ 2 H exchange data, however, show that helix a 3 , the C-terminal region of helix a 4 , and helix a 5 maintain low exchangeability of the amide protons in the LS 1 form. This finding most likely implies that these regions maintain some ordered non-covalent structure, in which the amide moieties are involved in H-bonds. Finally, a folding mechanism is proposed and discussed in terms of analogies and differences with the larger mitochondrial cytochrome c proteins. It is concluded that the thermodynamic stability of the region around the metal cofactor is determined by the chemical nature of the residues around the axial methionine residue.
Acta Biochimica Polonica, 2011
In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplastencoded cytochrome b 6 into the bacterial membrane. Cytochrome b 6 naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b 6 or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b 6 in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b 6 into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group . The results presented here show that cytochrome b 6 devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b 6 is possible, as long as the B and D helices bearing axial ligands to heme are present.
Biogenesis of mitochondrialc-type cytochromes
Journal of Bioenergetics and Biomembranes
Cytochromes e and c I are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochrome c is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochrome c~ is firmly anchored to the inner membrane. Both cytochrome c and c~ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochrome c and c 1 are drastically different. Cytochrome c I is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochrome c is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochrome c is synthesized as apocytochrome c without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heine group, catalyzed by cytochrome c heme lyase, is a requirement for transport. In summary, cytochrome c I import appears to follow a "conservative pathway" reflecting features of cytochrome c~ sorting in prokaryotic cells. In contrast, cytochrome c has "invented" a rather unique pathway which is essentially "non-conservative."
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2020
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