The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.     

Complex formation and electron transfer between mitochondrial cytochrome c and flavocytochrome c552 from Chromatium vinosum

Flavocytochrome c552 from Chromatium vinosum catalyzes the oxidation of sulfide to sulfur using a soluble c-type cytochrome as an electron acceptor. Mitochondrial cytochrome c forms a stable complex with flavocytochrome c552 and may function as an alternative electron acceptor in vitro. The recognition site for flavocytochrome c552 on equine cytochrome c has been deduced by differential chemical modification of cytochrome c in the presence and absence of flavocytochrome c552 and by kinetic analysis of the sulfide:cytochrome c oxidoreductase activity of m-trifluoromethylphenylcarbamoyl-lysine derivatives of cytochrome c. As with mitochondrial redox partners, interaction occurs around the exposed heme edge at the "front face" of cytochrome c. However, the domain recognized by flavocytochrome c552 seems to extend to the right of the heme edge, whereas the site of interaction with mitochondrial cytochrome c oxidase and reductase is more to the left. Km but not Vmax of the electron transfer reaction with mitochondrial cytochrome c increases with increasing ionic strength. The correlation of chemical modification and ionic strength dependence data indicates that the electrostatic interaction between the two hemoproteins involves fewer ionic bonds than that with other redox partners of cytochrome c.     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

Cytochromes c550, c552, and c1 in the Electron Transport Network of Paracoccus denitrificans: Redundant or Subtly Different in Function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.     

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

Mitochondrial cytochromes c and c(1) 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.     

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.     

Identification of a gene encoding a thioredoxin-like product necessary for cytochrome c biosynthesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

A Tn5-induced mutant of Rhizobium leguminosarum bv. viciae could not form nitrogen-fixing nodules on pea or vetch because of a lesion in electron transport to oxygen. The mutant lacked spectroscopically detectable cytochromes c and aa3. No proteins containing c-type cytochrome could be identified in the mutant by heme staining of proteins fractionated on polyacrylamide gels, indicating that the mutant was defective in maturation of all c-type cytochromes. The Tn5 mutation was determined to be located in a gene that was called cycY. The cycY gene product is homologous to the thioredoxin-like protein HelX involved in the assembly of c-type cytochromes in Rhodobacter capsulatus and to an open reading frame from a Bradyrhizobium japonicum gene cluster containing other genes involved in cytochrome c biogenesis. Our observations are consistent with CycY functioning as a thioredoxin that reduces cysteine residues in apocytochromes c before heme attachment.     

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.     

Rhodobacter capsulatus CycH: a bipartite gene product with pleiotropic effects on the biogenesis of structurally different c-type cytochromes.

While searching for components of the soluble electron carrier (cytochrome c2)-independent photosynthetic (Ps) growth pathway in Rhodobacter capsulatus, a Ps- mutant (FJM13) was isolated from a Ps+ cytochrome c2-strain. This mutant could be complemented to Ps+ growth by cycA encoding the soluble cytochrome c2 but was unable to produce several c-type cytochromes. Only cytochrome c1 of the cytochrome bc1 complex was present in FJM13 cells grown on enriched medium, while cells grown on minimal medium contained at various levels all c-type cytochromes, including the membrane-bound electron carrier cytochrome cy. Complementation of FJM13 by a chromosomal library lacking cycA yielded a DNA fragment which also complemented a previously described Ps- mutant, MT113, known to lack all c-type cytochromes. Deletion and DNA sequence analyses revealed an open reading frame homologous to cycH, involved in cytochrome c biogenesis. The cycH gene product (CycH) is predicted to be a bipartite protein with membrane-associated amino-terminal (CycH1) and periplasmic carboxyl-terminal (CycH2) subdomains. Mutations eliminating CyCH drastically decrease the production or all known c-type cytochromes. However, mutations truncating only its CycH2 subdomain always produce cytochrome c1 and affect the presence of other cytochromes to different degrees in a growth medium-dependent manner. Thus, the subdomain CycH1 is sufficient for the proper maturation of cytochrome c1 which is the only known c-type cytochrome anchored to the cytoplasmic membrane by its carboxyl terminus, while CycH2 is required for efficient biogenesis of other c-type cytochromes. These findings demonstrate that the two subdomains of CycH play different roles in the biogenesis of topologically distinct c-type cytochromes and reconcile the apparently conflicting data previously obtained for other species.     

Membrane-bound cytochromes in a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F

Cytoplasmic membranes were isolated from the cells of a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F and membrane-bound cytochromes were characterized. Redox difference spectra at 77 K revealed the presence of cytochromes with the alpha peaks at 552 and 556 nm while CO-binding difference spectra showed the presence of o-type cytochrome(s). Partial purification of the cytochromes demonstrated that the membranes contain cytochromes c550, c551, c556 and possibly d1 besides high molecular mass cytochrome c and cytochrome c3. It turned out that two kinds of novel CO-binding c-type cytochromes are present in the membrane. The membranes and a partially purified fraction showed weak ubiquinol-1 oxidase activity but no cytochrome c oxidase activity. Results suggest that D. vulgaris does not express the heme-copper terminal oxidase under our growth conditions in spite of the presence of the col gene, which is homologous to the gene of subunit I of the aa3-type oxidase.     

The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.     

MauG,a novel diheme protein required for tryptophan tryptophylquinone biogenesis

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits. None of these gene products have been previously isolated. One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH. A system was developed for the homologous expression of MauG in P. denitrificans. Its signal sequence was correctly processed, and it was purified from the periplasmic cell fraction. The protein contains two covalent c-type hemes, as predicted from the deduced sequence. EPR spectroscopy reveals that the protein as isolated possesses about equal amounts of one high-spin heme with axial symmetry and one low-spin heme with rhombic symmetry. The low-spin heme contains a major and minor component suggesting a small degree of heme heterogeneity. The high-spin heme and the major low-spin heme component each exhibit resonances that are atypical of c-type hemes and dissimilar to those reported for diheme cytochrome c peroxidases. MauG exhibited only very weak peroxidase activity when assayed with either c-type cytochromes or o-dianisidine as an electron donor. Fully reduced MauG was shown to bind carbon monoxide and could be reoxidized by oxygen. The relevance of these unusual properties of MauG is discussed in the context of its role in TTQ biogenesis.     

Dynamic features of a heme delivery system for cytochrome C maturation

In Escherichia coli, heme is delivered to cytochrome c in a process involving eight proteins encoded by the ccmABCDEFGH operon. Heme is transferred to the periplasmic heme chaperone CcmE by CcmC and from there to apocytochrome c. The role of CcmC was investigated by random as well as site-directed mutagenesis. Important amino acids were all located in periplasmic domains of the CcmC protein that has six membrane-spanning helices. Besides the tryptophan-rich motif and two conserved histidines, new residues were identified as functionally important. Mutants G111S and H184Y had a clear defect in CcmC-CcmE interaction, did not transfer heme to CcmE, and lacked c-type cytochromes. Conversely, mutants D47N, R55P, and S176Y were affected neither in interaction with nor in delivery of heme to CcmE but produced less than 10% c-type cytochromes. A strain carrying a CcmCE fusion had a similar phenotype, suggesting that CcmC is important not only for heme transfer to CcmE but also for its delivery to cytochrome c. Co-immunoprecipitation of CcmC with CcmF was not detectable although CcmE co-precipitated individually with CcmC and CcmF. This contradicts the idea of CcmCEF supercomplex formation. Our results favor a model that predicts CcmE to shuttle between CcmC and CcmF for heme delivery.     

Mutations in cytochrome assembly and periplasmic redox pathways in Bordetella pertussis

Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochrome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB- mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed.     

Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity.

A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis. P. denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes. In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type. The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104. The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced. Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media. Disruption of the hemA gene generated a P. denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism. The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion.     

The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex

The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.     

Characterization and redox properties of cytochrome c552 from Thermus thermophilus adsorbed on different self-assembled thiol monolayers, used to model the chemical environment of the redox partner

The structure of cytochrome c552 (Cyt-c552) from Thermus thermophilus shows many differences to other c-type cytochromes. The rich lysine domain close to the heme does not exist in this cytochrome, allowing us to postulate that the interaction with its redox partner must be different to the cytochrome c/cytochrome c oxidase interaction. We report a study of Cyt-c552 adsorbed on self-assembled monolayers (SAMs) of functionalized alkanethiols used to mimic the chemical properties of its redox partner (ba3-oxydase). Hydrophilic (-COOH), polar (-OH), hydrophobic (-CH3), and mixed (-OH/-CH3) SAMs grafted on roughened silver electrodes were characterized by X-ray photoelectron spectroscopy. Surface enhanced resonance Raman spectroscopy (SERRS) was employed to determine the structure and the redox properties (E degrees and number of transferred electron) of the heme of Cyt-c552 adsorbed on roughened silver electrodes coated by the different SAMs. The surface that most closely models the environment of the ba3-oxidase is a mixed SAM formed by 50% polar [Ag-(CH2)5-CH2OH] and 50% hydrophobic [Ag-(CH2)5-CH3] alkanethiols. Only the native form B1(6cLS) of Cyt-c552 is detected by SERRS when the protein is adsorbed on such a surface that promotes a protein orientation favorable for the electron transfer (number of transferred electron = 1). We shall discuss the differences and similarities of the electron-transfer mechanism of Cyt-c552 compared to cyt-c.     

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.