Cytochromes C Research Articles

Red complex I — Using cytochrome c550 from Bacillus subtilis as a fusion domain to study NADH:quinone oxidoreductase

Red complex I — Using cytochrome c550 from Bacillus subtilis as a fusion domain to study NADH:quinone oxidoreductase

A new CuZ active form in the catalytic reduction of N2O by nitrous oxide reductase from Pseudomonas nautica

The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.

Biochemical Studies of asoxF-Encoded Monomeric Flavoprotein Purified from the Green Sulfur BacteriumChlorobaculum tepidumThat Stimulatesin VitroThiosulfate Oxidation

In the green sulfur bacterium Chlorobaculum tepidum, three sulfur oxidizing enzyme system (Sox) proteins, SoxAXK, SoxYZ, and SoxB (the core TOMES, thiosulfate oxidizing multi-enzyme system) are essential to in vitro thiosulfate oxidation. We purified monomeric flavoprotein SoxF from this bacterium, which had sulfide dehydrogenase activity. SoxF enhanced the thiosulfate oxidation activity of the purified core TOMES with various cytochromes as electron acceptors to different degrees without any change in the affinity for thiosulfate. The apparent reaction rates with 50 microM- C. tepidum cytochrome c-554 were slightly higher than with horse-heart cytochrome c, and the addition of 0.5 microM- SoxF increased the rate by 92%. The rates with 50 microM- horse-heart cytochrome c and 50 muM- horse-heart cytochrome c plus 0.5 muM- cytochrome c-554 were increased by SoxF by 31% and 120% respectively. We conclude that SoxF mediates electron transfer between the components of core TOMES and externally added cytochromes.

Molecular Basis for Directional Electron Transfer

Biological macromolecules involved in electron transfer reactions display chains of closely packed redox cofactors when long distances must be bridged. This is a consequence of the need to maintain a rate of transfer compatible with metabolic activity in the framework of the exponential decay of electron tunneling with distance. In this work intermolecular electron transfer was studied in kinetic experiments performed with the small tetraheme cytochrome from Shewanella oneidensis MR-1 and from Shewanella frigidimarina NCIMB400 using non-physiological redox partners. This choice allowed the effect of specific recognition and docking to be eliminated from the measured rates. The results were analyzed with a kinetic model that uses the extensive thermodynamic characterization of these proteins reported in the literature to discriminate the kinetic contribution of each heme to the overall rate of electron transfer. This analysis shows that, in this redox chain that spans 23 A, the kinetic properties of the individual hemes establish a functional specificity for each redox center. This functional specificity combined with the thermodynamic properties of these soluble proteins ensures directional electron flow within the cytochrome even outside of the context of a functioning respiratory chain.

Organization of the electron transfer chain to oxygen in the obligate human pathogen Neisseria gonorrhoeae: roles for cytochromes c4 and c5, but not cytochrome c2, in oxygen reduction.

Although Neisseria gonorrhoeae is a prolific source of eight c-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes c(2), c(4), and c(5), encoded by the genes cccA, cycA, and cycB, respectively, have been investigated. Single mutations in genes for either cytochrome c(4) or c(5) resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible copy of the cycA or cycB gene. In contrast, a cccA mutant reduced oxygen slightly more rapidly than the parent, suggesting that cccA is expressed but cytochrome c(2) is not involved in electron transfer to cytochrome oxidase. The deletion of cccA increased the sensitivity of the cycB mutant to excess oxygen but decreased the sensitivity of the cycA mutant. Despite many attempts, a double mutant defective in both cytochromes c(4) and c(5) could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible cycB gene with deletions in both cycA and cycB was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome c(4) or c(5). These results define the gonococcal electron transfer chain to oxygen in which cytochromes c(4) and c(5), but not cytochrome c(2), provide alternative pathways for electron transfer from the cytochrome bc(1) complex to the terminal oxidase cytochrome cbb(3).

C-type cytochromes in the photosynthetic electron transfer pathways in green sulfur bacteria and heliobacteria

Green sulfur bacteria and heliobacteria are strictly anaerobic phototrophs that have homodimeric type 1 reaction center complexes. Within these complexes, highly reducing substances are produced through an initial charge separation followed by electron transfer reactions driven by light energy absorption. In order to attain efficient energy conversion, it is important for the photooxidized reaction center to be rapidly rereduced. Green sulfur bacteria utilize reduced inorganic sulfur compounds (sulfide, thiosulfate, and/or sulfur) as electron sources for their anoxygenic photosynthetic growth. Membrane-bound and soluble cytochromes c play essential roles in the supply of electrons from sulfur oxidation pathways to the P840 reaction center. In the case of gram-positive heliobacteria, the photooxidized P800 reaction center is rereduced by cytochrome c-553 (PetJ) whose N-terminal cysteine residue is modified with fatty acid chains anchored to the cytoplasmic membrane.

Effect of Salinity on Hydroxylamine Oxidation in a Marine Ammonia-Oxidizing Gammaproteobacterium, Nitrosococcus oceani strain NS58: Molecular and Catalytic Properties of Tetraheme Cytochrome c-554

Tetraheme cytochrome c-554 is a physiological electron acceptor of hydroxylamine oxidoreductase (HAO), a core enzyme of ammonia oxidation in chemoautotrophic nitrifiers. Here we report the purification of cytochrome c-554 from Nitrosococcus oceani strain NS58, a marine gammaproteobacterial ammonia-oxidizing bacterium. The NS58 cytochrome is a 25 kDa-protein having four hemes c. The absorption spectrum of the cytochrome showed peaks at 420 nm, 523 nm, and 554 nm, with shoulders at around 430 nm and 580 nm in the reduced state. In contrast to the highly basic counterpart from the betaproteobacterium Nitrosomonas europaea, the NS58 cytochrome c-554 was an acidic protein whose isoelectric point was 4.6. HAO was also purified, and the reaction with the NS58 cytochrome was found to be salt-tolerant. Compared with the activity observed in a non-salt solution, 60% of the activity remained in a saline concentration comparable to that of seawater.

Dissimilatory Fe (III) reduction by cytochrome c-552 in a thermophilic,obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6

A thermophilic, obligately chemolithoauto- trophic H2-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can utilize O2 or NO3- as an electron acceptor. Here we show that this bacterium can grow autotrophically by the reduction of Fe(III)-EDTA or Fe(III)-DTPA (diethylene-triamine-pentaacetic acid) as an electron acceptor, through oxidation of H2 as an electron donor. A 7.6 kDa c-type cytochrome was purified from soluble fraction as the iron reducing protein and the protein was identified as cytochrome c-552. A rate constant for the reduction of Fe(III)-EDTA by the cytochrome was kinetically determined to be 1.73 x 107 s-1 by stopped-flow spectrophotometry, which is more than 20-fold higher than the constants of other iron reductases. The redox potential was measured as E = +0.27 V (vs NHE [Normal Hydrogen Electrode]), which is high enough to reduce Fe(III)-EDTA. These results suggest that cytochrome c-552 works as a terminal electron donor for Fe(III) in the periplasmic space.

3P064 高時間分解能一分子測定による緑膿菌由来シトクロムcの変性状態内の運動観察(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

3P064 高時間分解能一分子測定による緑膿菌由来シトクロムcの変性状態内の運動観察(蛋白質-物性(安定性,折れたたみなど),第48回日本生物物理学会年会)

Rational engineering of Thermus thermophilus cytochrome c552 to a thermally tolerant artificial peroxidase

In a previous study, we constructed a prototype thermally tolerant artificial peroxidase from an electron transfer protein, cytochrome c(552) from Thermus thermophilus, and demonstrated that engineering of proteins from thermophiles could be a promising methodology to produce artificial enzymes for practical use. In the present study, further improvement of the prototype (the V49D/M69A mutant) in enzymatic activity and thermal tolerance has been achieved by successive modifications based on detailed analyses of an active intermediate formed in the peroxidase reaction. Spectroscopic studies revealed that the major active intermediate of V49D/M69A was an oxo-ferryl heme with a protein radical predominantly localized on Tyr45. The magnetic power saturation measurement in EPR studies showed little magnetic coupling between the oxo-ferryl heme and the tyrosyl radical. This result indicated that the oxo-ferryl heme and the tyrosyl radical served as isolated oxidants. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate could be a precursor to heme degradation by the reaction with H(2)O(2). Replacement of Tyr45 with phenylalanine on V49D/M69A resulted in delocalization of the radical over the protein and increased magnetic coupling between the oxo-ferryl heme and the protein radical in the intermediate. The stronger magnetic coupling between the oxo-ferryl heme and the radical was achieved by replacement of Tyr45 with tryptophan, which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. The protein radical intermediates of the Tyr45 mutants exhibited relatively higher reactivity to an organic substrate than H(2)O(2) in comparison to the basal mutant, V49D/M69A, which was preferable to suppress the heme degradation process. This was reflected in the improved enzymatic activity and thermal tolerance observed for the Tyr45 mutants in the peroxidase reaction.

1P028 Intron Positions and Module Boundaries of Cytochrome C6 and its Function(Protein:Structure & Function,The 48th Annual Meeting of the Biophysical Society of Japan)

1P028 Intron Positions and Module Boundaries of Cytochrome C6 and its Function(Protein:Structure & Function,The 48th Annual Meeting of the Biophysical Society of Japan)

X-Ray Structure Determination of Isocytochrome C2 from the Photosynthetic Bacterium Rhodobacter sphaeroides

In cytochrome c2-deficient strains of the photosynthetic bacterium, Rhodobacter sphaeroides, a related c-type cytochome, isocytochome c2 replaces cytochrome c2 as the secondary electron donor to the photo-oxidized bacteriochlorophyll dimer in the reaction center. We will describe the x-ray structure determination of isocytochrome c2 at 2.2 Å from Rb sphaeroides.Analysis of the x-ray diffraction data indicated the crystals belong to space group P3(2) with unit cell dimensions a=b=56.0 Å c=90.5 Å and are merohederally twinned.

Mutation analysis of the interaction of B-type cytochrome c oxidase with its natural substrate cytochrome c-551

Heme-copper oxidases in the respiratory chain are classified into three subfamilies: A-, B- and C-types. Cytochrome bo(3)-type cytochrome c oxidase from thermophilic Bacillus is a B-type oxidase that is thought to interact with cytochrome c through hydrophobic interactions. This is in contrast to A-type oxidases, which bind cytochrome c molecules primarily through electrostatic forces between acidic residues in the oxidase subunit II and basic residues within cytochromes. In order to investigate the substrate-binding site in cytochrome bo(3), eight acidic residues in subunit II were mutated to corresponding neutral residues and enzymatic activity was measured using cytochrome c-551 from closely related Bacillus PS3. The mutation of E116, located at the interface to subunit I, decreased the k(cat) value most prominently without affecting the K(m) value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the Cu(A) site, largely affected both values, suggesting that it is important for both electron transfer and substrate binding. The mutation of D49 and E84 did not affect enzyme kinetic parameters, but the mutation of E64, E66 and E68 lowered the affinity of cytochrome bo(3) for cytochrome c-551 without affecting the k(cat) value. These three residues are located at the front of the hydrophilic globular domain and distant from the Cu(A) site, suggesting that these amino acids compose an acidic patch for a second substrate-binding site. This is the first report on site-directed mutagenesis experiments of a B-type heme-copper oxidase.

Structural characterization of a family of cytochromes c7 involved in Fe(III) respiration by Geobacter sulfurreducens

Periplasmic cytochromes c7 are important in electron transfer pathway(s) in Fe(III) respiration by Geobacter sulfurreducens. The genome of G. sulfurreducens encodes a family of five 10-kDa, three-heme cytochromes c7. The sequence identity between the five proteins (designated PpcA, PpcB, PpcC, PpcD, and PpcE) varies between 45% and 77%. Here, we report the high-resolution structures of PpcC, PpcD, and PpcE determined by X-ray diffraction. This new information made it possible to compare the sequences and structures of the entire family. The triheme cores are largely conserved but are not identical. We observed changes, due to different crystal packing, in the relative positions of the hemes between two molecules in the crystal. The overall protein fold of the cytochromes is similar. The structure of PpcD differs most from that of the other homologs, which is not obvious from the sequence comparisons of the family. Interestingly, PpcD is the only cytochrome c7 within the family that has higher abundance when G. sulfurreducens is grown on insoluble Fe(III) oxide compared to ferric citrate. The structures have the highest degree of conservation around “heme IV”; the protein surface around this heme is positively charged in all of the proteins, and therefore all cytochromes c7 could interact with similar molecules involving this region. The structures and surface characteristics of the proteins near the other two hemes, “heme I” and “heme III”, differ within the family. The above observations suggest that each of the five cytochromes c7 could interact with its own redox partner via an interface involving the regions of heme I and/or heme III; this provides a possible rationalization for the existence of five similar proteins in G. sulfurreducens.

Enzymatic activity of the alternative complex III as a menaquinol:auracyanin oxidoreductase in the electron transfer chain of Chloroflexus aurantiacus

The surprising lack of the cytochrome bc 1 complex in the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus suggests that a functional replacement exists to link the cyclic electron transfer chain. Earlier work identified the alternative complex III (ACIII) as a substitute of cytochrome bc 1 complex. Herein, the enzymatic activity of ACIII is studied. The results strongly support the view that the ACIII functions as menaquinol:auracyanin oxidoreductase in the C. aurantiacus electron transfer chain. Among all the substrates tested, auracyanin is the most efficient electron acceptor of ACIII, suggesting that ACIII directly transfers the electron to auracyanin instead of cytochrome c-554. The lack of sensitivity to common inhibitors of the cytochrome bc 1 complex indicates a different catalytic mechanism for the ACIII complex.

Cytochrome c4 Can Be Involved in the Photosynthetic Electron Transfer System in the Purple Bacterium Rubrivivax gelatinosus

Three periplasmic electron carriers, HiPIP and two cytochromes c8 with low- and high-midpoint potentials, are present in the purple photosynthetic bacterium Rubrivivax gelatinosus. Comparison of the growth rates of mutants lacking one, two, or all three electron carrier proteins showed that HiPIP is the main electron donor to the photochemical reaction center and that high-potential cytochrome c8 plays a subsidiary role in the electron donation in photosynthetically growing cells. However, the triple deletion mutant was still capable of photosynthetic growth, indicating that another electron donor could be present. A new soluble cytochrome c, which can reduce the photooxidized reaction center in vitro, was purified. Based on amino acid sequence comparisons to known cytochromes, this cytochrome was identified as a diheme cytochrome c of the family of cytochromes c4. The quadruple mutant lacking this cytochrome and three other electron carriers showed about three times slower growth than the triple mutant under photosynthetic growth conditions. In conclusion, cytochrome c4 can function as a physiological electron carrier in the photosynthetic electron transport chain in R. gelatinosus.

Cytochrome c551 Is a Mediator of Electron Transfer between Copper-Containing Nitrite Reductase and Azurin in a Denitrifying Bacterium, Achromobacter xylosoxidans

Abstract Cu-containing nitrite reductase from a denitrifying bacterium, Achromobacter xylosoxidans GIFU1051 (the organism formerly known as Alcaligenes xylosoxidans), can accept an electron from three possible electron donors (cytochrome c551 and two azurins). We have kinetically demonstrated that cytochrome c551 functions as a mediator in the electron flow processes between the enzyme and two azurins.

Effect of the Redox-Dependent Ionization State of the Heme Propionic Acid Side Chain on the Entropic Contribution to the Redox Potential of Pseudomonas aeruginosa Cytochrome c551

The thermodynamic properties of the redox potentials (E(m)) of Pseudomonas aeruginosa cytochrome c(551) (PA) and its mutants possessing a variety of pK(a) values for the heme 17-propionic acid side chain, which ranged from approximately 5 to approximately 8, have been investigated to elucidate the role of ionization of the heme side chain in the E(m) control. Since the pK(a) values of the heme 17-propionic acid side chains of the oxidized and reduced forms of PA are 5.9 +/- 0.2 and 7.0 +/- 0.2, respectively [Takayama, S. J., Mikami, S., Terui, N., Mita, H., Hasegawa, J., Sambongi, Y., and Yamamoto, Y. (2005) Biochemistry 44, 5488-5494], the ionization state of the heme 17-propionic acid side chain at physiological pH depends on the oxidation state of the protein. This redox-dependent ionization state of the heme 17-propionic acid side chain was found to have a large effect on the entropic contribution (DeltaS) to the E(m) value. The magnitude of the E(m) control through the DeltaS value due to the redox-dependent ionization state of the heme 17-propionic acid side chain was shown to be about 170 mV and hence is considerably larger than that through the enthalpic contribution (DeltaH) to the E(m) value due to stabilization of the cationic ferriheme in the oxidized protein through partial neutralization of its positive charge by the heme 17-propionate side chain, i.e., about 60 mV [Takayama, S. J., Mikami, S., Terui, N., Mita, H., Hasegawa, J., Sambongi, Y., and Yamamoto, Y. (2005) Biochemistry 44, 5488-5494]. The present study revealed that the heme 17-propionic acid side chain of the protein plays a pivotal role in the E(m) control of the protein.

Physiological role and redox properties of a small cytochrome c5, cytochrome c-552, from alkaliphile, Pseudomonas alcaliphila AL15-21T

Cytochrome c-552 from Pseudomonas alcaliphila AL15-21(T), which is a small cytochrome c(5) from Pseudomonas spp., was first purified and characterized in our previous study. Although it has been found that cytochrome c-552 is induced at a high pH under air-limited condition, the physiological role of this cytochrome c has not been clarified yet. Therefore, to understand its physiological role, further characterization of this cytochrome expressed in Escherichia coli was performed. The yield of the recombinant protein reached 2.8 mg/l of culture, which was 76.4-fold larger than that of native cells. Analytical data of the recombinant protein exactly agreed with that of native cytochrome c-552. The recombinant cytochrome c-552 was oxidized by partially purified cb-type cytochrome c oxidase from P. alcaliphila AL15-21(T) at a rate of 9.6 mu mol min(-1) mg oxidase(-1). Unlike reported cytochromes c from other Pseudomonas spp., the E degrees ' values between pHs 5.0 and 10.0 were nearly unchanged. Cytochrome c-552 oxidized very slowly at pHs 8.0 (6.1 x 10(-4) h(-1)), 9.0 (1.4 x 10(-3) h(-1)) and 10.0 (1.6 x 10(-3) h(-1)), whereas it oxidized more rapidly at pH 7.0 (2.5 x 10(-3) h(-1)). On the other hand, horse heart cytochrome c showed higher oxidation rates at pHs 6.0-10.0 than cytochrome c-552. It is considered that the high electron-retaining ability of cytochrome c-552 at high pHs is important for its physiological function in the environmental adaptation of this bacteria for superior growth at high pHs under air-limited conditions.

Dynamics of Adaptive Microevolution of HypermutablePseudomonas aeruginosaduring Chronic Pulmonary Infection in Patients with Cystic Fibrosis

In patients with cystic fibrosis (CF), the emergence of hypermutable Pseudomonas aeruginosa drives the selection of P. aeruginosa variants that are efficiently adapted to the inflamed lungs of these patients. To provide a detailed survey of adaptive changes in the physiology of P. aeruginosa during chronic lung infection in patients with CF. We performed a comparative proteome and transcriptome analysis of sequential, isogenic isolates recovered over a period of 3-5 years from 3 selected patients with CF. The isolates analyzed included both those with high mutation rates and defects in their methyl-directed mismatch repair system (hereafter, "mutators") and those without such changes (hereafter, "nonmutators"). In addition to attenuation of virulence, the P. aeruginosa adaptation process predominantly affects metabolic pathways. In mutator isolates recovered from patients with end-stage CF lung disease, we observed increases in the transcripts of genes or proteins involved in the metabolism of fatty acids and amino acids and the generation of energy. Of particular interest is the increased expression of genes involved in the following pathways and processes: (1) the anaerobic arginine-deiminase pathway, (2) anaerobic respiration (e.g., nitrate-uptake protein OprF, azurin, and cytochrome c551 peroxidase), (3) microaerobic respiration (e.g., cytochrome oxidase cbb3), and (4) the tricarboxylic acid cycle and glyoxylate shunt. Strikingly, increased transcription of the anaerobic regulator gene anr correlates with the up-regulation of ANR-dependent genes. These changes indicate an adaptive shift toward constitutive expression of genes required for growth under the nutritional and microaerobic conditions created by suppurative secretions in the lungs of patients with CF. In addition, these results provide important clues about the persistence strategies used by P. aeruginosa during progressive CF lung disease.