Biomedical subjects

James W A Allen

Publications and source records attributed to James W A Allen.

14 recordsLinked to original sources

PubMed2006

A variant System I for cytochrome c biogenesis in archaea and some bacteria has a novel CcmE and no CcmH.

C-type cytochromes are characterized by post-translational covalent attachment of heme to thiols that occur in a Cys-Xxx-Xxx-Cys-His motif. Three distinct biogenesis systems are known for this heme attachment. Archaea are now shown to contain a significantly modified form of cytochrome c maturation System I (the Ccm system). The most notable adaptation relative to the well-studied apparatus from proteobacteria and plants is a novel form of the heme chaperone CcmE, lacking the highly conserved histidine that covalently binds heme and is essential for function in Escherichia coli. In most archaeal CcmEs this histidine, normally found in a His-Xxx-Xxx-Xxx-Tyr motif, is replaced by a cysteine residue that occurs in a Cys-Xxx-Xxx-Xxx-Tyr motif. The CcmEs from two halobacteria contain yet another form of CcmE, having HxxxHxxxH approximately corresponding in alignment to the H/CxxxY motif. The CxxxY-type of CcmE is, surprisingly, also found in some bacterial genomes (including Desulfovibrio species). All of the modified CcmEs cluster together in a phylogenetic tree, as do other Ccm proteins from the same organisms. Significantly, CcmH is absent from all of the complete archaeal genomes we have studied, and also from most of the bacterial genomes that have CxxxY-type CcmE.

Amino Acid Sequence↗

PubMed2005

Why isn't 'standard' heme good enough for c-type and d1-type cytochromes?

This perspective seeks to discuss why biology often modifies the fundamental iron-protoporphyrin IX moiety that is the very versatile cofactor of many heme proteins. A very common modification is the attachment of this cofactor via covalent bonds to two (or rarely one) sulfur atoms of cysteine residue side chains. This modification results in c-type cytochromes, which have diverse structures and functions. The covalent bonds are made in different ways depending on the cell type. There is little understanding of the reasons for this complexity in assembly routes but proposals for the rationale behind the covalent modification are presented. In contrast to the widespread c-type cytochromes, the d1 heme is restricted to a single enzyme, the cytochrome cd1 nitrite reductase that catalyses the one-electron reduction of nitrite to nitric oxide. This is an extensively derivatised heme; a comparison is drawn with another type of respiratory nitrite reductase in which the active site is a c-type heme, but the product ammonia.

Antibodies, Monoclonal↗

PubMed2005

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.

Amino Acid Substitution↗

PubMed2004

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.

Amino Acid Motifs↗

PubMed2004

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase.

The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.

Catalysis↗

PubMed2004

C-type cytochrome formation: chemical and biological enigmas.

C-type cytochromes are proteins that are essential for the life of virtually all organisms. They characteristically contain heme that is covalently attached via thioether bonds to two cysteines in the protein. In this Account, we describe the challenging chemistry of thioether bond formation and the surprising variety of biogenesis systems that exist in nature to perform the difficult posttranslational heme attachment process. We show what insight has been gained into the various biogenesis systems from in vitro and in vivo experiments and highlight some forthcoming challenges in this field.

Animals↗

PubMed2003

Variation of the axial haem ligands and haem-binding motif as a probe of the Escherichia coli c-type cytochrome maturation (Ccm) system.

Cytochromes c are typically characterized by the covalent attachment of haem 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 haem is attached to the polypeptide by the periplasmically functioning cytochrome c maturation (Ccm) proteins. Exceptionally, Hydrogenobacter thermophilus cytochrome c552 can be expressed as a stable holocytochrome both in the cytoplasm of Escherichia coli in an apparently uncatalysed reaction and also in the periplasm in a Ccm-mediated reaction. In the present study we show that a Met60-->Ala variant of c552, which does not have the usual distal methionine ligand to the haem iron of the mature cytochrome, can be made in the periplasm by the Ccm system. However, no holocytochrome could be detected when this variant was expressed cytoplasmically. These data highlight differences between the two modes of cytochrome c assembly. In addition, we report investigations of haem attachment to cytochromes altered to have the special Cys-Trp-Ser-Cys-Lys haem-binding motif, and Cys-Trp-Ser-Cys-His and Cys-Trp-Ala-Cys-His analogues, of the active-site haem of nitrite reductase NrfA.

Amino Acid Motifs↗

PubMed2003

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 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.

Amino Acid Motifs↗

PubMed2003

C-type cytochromes: diverse structures and biogenesis systems pose evolutionary problems.

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.

Amino Acid Motifs↗

PubMed2003

Structure and kinetic properties of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with the d1 heme active site ligand tyrosine 25 replaced by serine.

The 1.4-A crystal structure of the oxidized state of a Y25S variant of cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr(25), a ligand via its hydroxy group to the iron of the d(1) heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser(25) side chain is seen in two positions in the d(1) heme pocket with relative occupancies of approximately 7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser(25) side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr(25) is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d(1) heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.

Binding Sites↗

PubMed2002

A novel, kinetically stable, catalytically active, all-ferric, nitrite-bound complex of Paracoccus pantotrophus cytochrome cd1.

The oxidized form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase, as isolated, has bis-histidinyl co-ordination of the c haem and His/Tyr co-ordination of the d(1) haem. On reduction, the haem co-ordinations change to His/Met and His/vacant respectively. If the latter form of the enzyme is reoxidized, a conformer is generated in which the ferric c haem is His/Met co-ordinated; this can revert to the 'as isolated' state of the enzyme over approx. 20 min at room temperature. However, addition of nitrite to the enzyme after a cycle of reduction and reoxidation produces a kinetically stable, all-ferric complex with nitrite bound to the d(1) haem and His/Met co-ordination of the c haem. This complex is catalytically active with the physiological electron donor protein pseudoazurin. The effective dissociation constant for nitrite is 2 mM. Evidence is presented that d(1) haem is optimized to bind nitrite, as opposed to other anions that are commonly good ligands to ferric haem. The all-ferric nitrite bound state of the enzyme could not be generated stoichiometrically by mixing nitrite with the 'as isolated' conformer of cytochrome cd(1) without redox cycling.

Catalysis↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Motifs↗

PubMed2001

Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme.

Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c. At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1). Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins. There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor. A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported. Important implications for the enzymology of P. pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies.

Animals↗