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Stuart J Ferguson

Publications and source records attributed to Stuart J Ferguson.

At least 37 records · Page 2Linked to original sources
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↗
PubMed2003

A mutant of Paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase.

In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative electron carriers from the cytochrome bc(1) complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c(550). The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c(550) or both these proteins. We concluded that pseudoazurin and cytochrome c(550) are the alternative electron mediator proteins between the cytochrome bc(1) complex and the cytochrome cd(1)-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

Anaerobiosis↗
PubMed2002

Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.

NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the cleavage site showed it was between two equally sized di-haem domains predicted from sequence analysis.

Amino Acid Sequence↗
PubMed2002

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.

Animals↗
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

The CcmE protein of the c-type cytochrome biogenesis system: unusual in vitro heme incorporation into apo-CcmE and transfer from holo-CcmE to apocytochrome.

Three key steps of cytochrome c biogenesis in many Gram-negative bacteria, the uptake of heme by the heme chaperone CcmE, the covalent attachment of heme to CcmE, and its subsequent release from CcmE to an apocytochrome c, have been achieved in vitro. apo-CcmE from Escherichia coli preferentially bound to ferric, with high affinity (K(d), 200 nM), rather than ferrous heme. The preference for ferric heme was confirmed by competition with 8-anilino-1-naphthalenesulfonate, which bound to a hydrophobic pocket in apo-CcmE. Reduction under certain conditions of the ferric heme-CcmE complex, which has characteristics of a b-type cytochrome, resulted in covalent attachment of heme to the protein. The resulting in vitro-produced holo-CcmE was identical to the in vivo-produced holo-CcmE, proving that unmodified Fe-protoporphyrin IX is incorporated into CcmE. Only noncovalent binding of mesoheme to CcmE was observed, thus implicating at least one vinyl group in covalent binding of heme to CcmE. Heme transferred in vitro from holo-CcmE to apocytochrome c, provided the heme was reduced. The necessity for reduced holo-CcmE might explain the role of the heme chaperone, i.e., prevention of reaction of ferric heme with apocytochrome and thus avoidance of incorrect side products. In addition, an AXXAH mutant of the CXXCH binding motif in the apocytochrome c was unable to accept heme from holo-CcmE. These in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during c-type cytochrome maturation in many species of bacteria in vivo.

Apoenzymes↗
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↗
PubMed2002

Mo(V) co-ordination in the periplasmic nitrate reductase from Paracoccus pantotrophus probed by electron nuclear double resonance (ENDOR) spectroscopy.

The first electron nuclear double resonance (ENDOR) study of a member of the Mo-bis-molybdopterin guanine dinucleotide family of molybdoenzymes is presented, using the periplasmic nitrate reductase from Paracoccus pantotrophus. Rapid freeze-quenched time-resolved EPR revealed that during turnover the intensity of a Mo(V) EPR signal known as High-g [resting] increases. This signal is split by two interacting protons that are not solvent-exchangeable. X-band proton-ENDOR analysis resolved broad symmetrical resonance features that arose from four classes of protons weakly coupled to the Mo(V). Signals from two of these were lost upon exchange into deuterated buffer, suggesting that they may originate from OH(-) or H(2)O groups. One of these signals was also lost when the enzyme was redox-cycled in the presence of azide. Since these protons are very weakly coupled OH/H(2)O groups, they are not likely to be ligated directly to the Mo(V). This suggests that protonation of a Mo(VI)zO group does not occur on reduction to Mo(V), but most probably accompanies reduction of Mo(V) to Mo(IV). A resonance feature from a more strongly coupled proton, that was not lost following exchange into deuterated buffer, could also be resolved at 22-24 MHz. The anisotropy of this feature, determined from ENDOR spectra collected at a range of field positions, indicated a Mo-proton distance of approx. 3.2 A, consistent with this being one of the beta-methylene protons of a Mo-Cys ligand.

Amino Acid Sequence↗
PubMed2002

Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus.

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane-bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane-bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.

Amino Acid Sequence↗
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↗
PubMed2000

Control of periplasmic nitrate reductase gene expression (napEDABC) from Paracoccus pantotrophus in response to oxygen and carbon substrates.

The napEDABC operon of Paracoccus pantotrophus encodes a periplasmic nitrate reductase (NAP), together with electron-transfer components and proteins required for the synthesis of a fully functional enzyme. Previously, it had been shown that high NAP activity was observed when P. pantotrophus was grown aerobically on highly reduced carbon sources such as butyrate or caproate, but not when cultured on more oxidized substrates such as succinate or malate. The enzyme is not present to any extent when the organism is grown anaerobically under denitrifying conditions, regardless of the carbon source. Transcriptional analyses of the nap operon have now identified two initiation sites which were differentially regulated in response to the carbon source, with expression being maximal when cells were grown aerobically with butyrate. Analysis of a P. pantotrophus mutant (M6) deregulated for NAP activity identified a single C-->A transversion in a heptameric inverted-repeat sequence that partially overlapped the proximal promoter. Transcription analysis of this mutant revealed that expression of nap was completely derepressed under all growth conditions examined. Taken together, these findings indicate that nap transcription is negatively regulated during anaerobiosis, such that expression is restricted to aerobic growth, but only when the carbon source is highly reduced.

Anaerobiosis↗
PubMed2000

Transcriptional analysis of the nirS gene, encoding cytochrome cd1 nitrite reductase, of Paracoccus pantotrophus LMD 92.63.

The gene for cytochrome cd1 nitrite reductase of Paracoccus pantotrophus, a protein of known crystal structure, is nirS. This gene is shown to be flanked by genes previously recognized in other organisms to encode proteins involved in the control of its transcription (nirI) and the biosynthesis of the d1 cofactor (nirE). Northern blot analysis has established under anaerobic conditions that a monocistronic transcript is produced from nirS, in contrast to observations with other denitrifying bacteria in which arrangement of flanking genes is different and the messages produced are polycistronic. The lack of a transcript under aerobic conditions argues against a role for cytochrome cd1 in the previously proposed aerobic denitrification pathway in Pa. pantotrophus. A putative rho-independent transcription termination sequence immediately following nirS, and preceding nirE, can be identified. The independent transcription of nirS and nirE indicates that it should be possible to produce site-directed mutants of nirS borne on a plasmid in a nirS deletion mutant. The transcript start point for nirS has been determined by two complementary techniques, 5'-RACE (Rapid amplification of cDNA 5' ends) and primer extension. It is 29 bp upstream of the AUG of nirS. An anaerobox, which presumably binds Nnr, is centred a further 41.5 bp upstream of the transcript start. No standard sigma70 DNA sequence motifs can be identified, but a conserved sequence (T-T-GIC-C-G/C-G/C) can be found in approximately the same position (-16) upstream of the transcript starts of nirS and nirI, whose products are both involved in the conversion of nitrite to nitric oxide.

Base Sequence↗
PubMed1999

Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB.

Each of the Paracoccus denitrificans genes in the c-type cytochrome biogenesis gene cluster ccmABCDG, plus the two flanking genes ORF117 and hisH, were individually disrupted by omega insertion. Resultant phenotypes were restored to the wild-type by complementation from a set of plasmids. All of the ccm genes, but neither ORF117 nor hisH, were required for c-type cytochrome biogenesis; only ccmG was also implicated in the biosynthesis of cytochrome aa3. Disruption of ccmC or ccmG resulted in failure to grow on rich media, but disruption of ccmA, ccmB or ccmD did not. The ccmC mutant, but not the ccmA, ccmB or ccmD mutants, also exhibited the increased sensitivity to growth inhibition by oxidized thiol compounds previously observed for the ccmG mutant. In contrast to the ccmG mutant, however, growth of the ccmC mutant on rich media could not be restored by DTT. Siderophore biosynthesis and/or secretion by P. denitrificans was also attenuated by disruption of ccmC and ccmG but not of ccmA, ccmB or ccmD. These results indicate that CcmC can function independently of CcmA, CcmB and CcmD despite other evidence that these gene products form an ATP-binding cassette (ABC)-type-transporter with the subunit composition (CcmA)2-CcmB-CcmC or (CcmA)2-CcmB-CcmC-CcmD, and also suggest a possible link between the functions of CcmC and CcmG.

ATP-Binding Cassette Transporters↗
PubMed1998

Identification of the contiguous Paracoccus denitrificans ccmF and ccmH genes: disruption of ccmF, encoding a putative transporter, results in formation of an unstable apocytochrome c and deficiency in siderophore production.

Apocytochrome C550 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c-type cytochromes, produces reduced levels of siderophores and carries a Tn5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondrial haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates at least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli gamma-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-deficient mutant, the apo form of P. denitrificans cytochrome C550 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.

Amino Acid Sequence↗
PubMed1997

Disruption of the Pseudomonas aeruginosa dipZ gene, encoding a putative protein-disulfide reductase, leads to partial pleiotropic deficiency in c-type cytochrome biogenesis.

The Pseudomonas aeruginosa dipZ gene has been cloned and sequenced. Whereas disruption of Escherichia coli dipZ (dsbD), the hydrophilic C-terminal domain of which has been deduced to be periplasmic and to function as a protein-disulfide reductase, leads to the absence of c-type cytochromes, disruption of P. aeruginosa dipZ attenuated, but did not abolish, holo-c-type cytochrome biosynthesis. Comparison of the P. aeruginosa DipZ sequence with three other DipZ sequences indicated that there are not only two conserved cysteine residues in the C-terminal hydrophilic domain, but also two more in the central highly hydrophobic domain. The latter would be located toward the centre of two of the eight membrane-spanning alpha-helices predicted to compose the hydrophobic central domain of DipZ. Both these cysteine residues, plus other transmembrane helix residues, notably prolines and glycines, are also conserved in a group of membrane proteins, related to Bacillus subtilis CcdA, which lack the N- and C-terminal hydrophilic domains of the DipZ proteins. It is proposed that DipZ of P. aeruginosa and other organisms transfers reducing power from the cytoplasm to the periplasm through reduction and reoxidation of an intramembrane disulfide bond, or other mechanism involving these cysteine residues, and that this function can also be performed by B. subtilis CcdA and other CcdA-like proteins. The failure of dipZ disruption to abolish c-type cytochrome synthesis in P. aeruginosa suggests that, in contrast to the situation in E. coli, the absence of DipZ can be compensated for by one or more other proteins, for example a CcdA-like protein acting in tandem with one or more thioredoxin-like proteins.

Amino Acid Sequence↗