James W. A. Allen

In vitro formation of a c-type cytochrome

C-type cytochromes are essential for almost all organisms; they are characterized by the covalent attachment of heme to protein through two thioether bonds to a Cys-Xaa-Xaa-Cys-His peptide motif. Here we show, contrary to opinion of 30 years standing, that a c-type cytochrome can form from heme and apoprotein in vitro under mild conditions and in the absence of any biosynthesis apparatus. This reaction occurs provided formation of a disulfide bond within the Cys-Xaa-Xaa-Cys-His motif is avoided. There are important implications for understanding in vivo cytochrome c assembly.

Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway

The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif. Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH). There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms. The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1. In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus. Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli. However, a CXXCH variant was matured by the E. coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T. brucei cytochrome c can accommodate a second cysteine in a CXXCH motif. The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space. Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system.

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

Mitochondrial cytochromes c and c1 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.

A Cytochrome b562 Variant with a c-Type Cytochrome CXXCH Heme-binding Motif as a Probe of the Escherichia coli Cytochrome c Maturation System

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° 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.

Cytochrome c assembly: A tale of ever increasing variation and mystery?

Formation of cytochromes c requires a deceptively simple post-translational modification, the formation of two thioether bonds (or rarely one) between the thiol groups of two cysteine residues found in a CXXCH motif (with some occasional variations) and the vinyl groups of heme. There are three partially characterised systems for facilitating this post-translational modification; within these systems there is also variation. In addition, there are clear indications for two other distinct systems. Here some of the current issues in understanding the systems are analysed.

Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite

Cytochrome cd 1 is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d 1 heme. We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d 1-NO complex (formallyd 1Fe(II)-NO+) as a product of the reaction between fully reduced Paracoccus pantotrophuscytochrome cd 1 and nitrite, in the absence of excess reductant. The Fe-14NO ν(NO) stretching mode is observed at 1913 cm−1 with the corresponding Fe-15NO band at 1876 cm−1. Thisd 1 heme-NO complex is still readily observed after 15 min. EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d 1 heme, and a cFe(III)d 1Fe(II)-NO complex is formed. Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III)d 1Fe(II)-NO and 45% cFe(II)d 1Fe(II)-NO+. No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme. Implications for the mechanism of P. pantotrophuscytochrome cd 1 are discussed.

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 c1. 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 c1 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.

The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

Distinctive biochemistry in the trypanosome mitochondrial intermembrane space suggests a model for stepwise evolution of the MIA pathway for import of cysteine‐rich proteins

Mia40‐dependent disulphide bond exchange is used by animals, yeast, and probably plants for import of small, cysteine‐rich proteins into the mitochondrial intermembrane space (IMS). During import, electrons are transferred from the imported substrate to Mia40 then, via the sulphydryl oxidase Erv1, into the respiratory chain. Curiously, however, there are protozoa which contain substrates for Mia40‐dependent import, but lack Mia40. There are also organisms where Erv1 is present in the absence of respiratory chain components. In accommodating these and other relevant observations pertaining to mitochondrial cell biology, we hypothesise that the ancestral IMS import pathway for disulphide‐bonded proteins required only Erv1 (but not Mia40) and identify parasites in which O2 is the likely physiological oxidant for Erv1.

A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis.

Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates. Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle. Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis. A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with ‘switched’ axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine. This activated conformer returns over 20 min at 25 °C to the state previously observed for oxidized ‘as isolated’ cd1, which is catalytically inactive towards the same substrates.