Cytochromes C Research Articles

Comparison of the backbone dynamics of wild-type Hydrogenobacter thermophilus cytochrome c(552) and its b-type variant.

Cytochrome c552 from the thermophilic bacterium Hydrogenobacter thermophilus is a typical c-type cytochrome which binds heme covalently via two thioether bonds between the two heme vinyl groups and two cysteine thiol groups in a CXXCH sequence motif. This protein was converted to a b-type cytochrome by substitution of the two cysteine residues by alanines (Tomlinson and Ferguson in Proc Natl Acad Sci USA 97:5156–5160, 2000a). To probe the significance of the covalent attachment of the heme in the c-type protein, 15N relaxation and hydrogen exchange studies have been performed for the wild-type and b-type proteins. The two variants share very similar backbone dynamic properties, both proteins showing high 15N order parameters in the four main helices, with reduced values in an exposed loop region (residues 18–21), and at the C-terminal residue Lys80. Some subtle changes in chemical shift and hydrogen exchange protection are seen between the wild-type and b-type variant proteins, not only for residues at and neighbouring the mutation sites, but also for some residues in the heme binding pocket. Overall, the results suggest that the main role of the covalent linkages between the heme group and the protein chain must be to increase the stability of the protein.

Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c 551 (PA cyt c 551), and Hydrogenobacter thermophilus cytochrome c 552 (HT cyt c 552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c 552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c 551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c 551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

Bidirectional Photoinduced Electron Transfer in Ruthenium(II)-Tris-bipyridyl-Modified PpcA, a Multi-heme c-Type Cytochrome from Geobacter sulfurreducens.

PpcA, a tri-heme cytochrome c7 from Geobacter sulfurreducens, was investigated as a model for photosensitizer-initiated electron transfer within a multi-heme "molecular wire" protein architecture. Escherichia coli expression of PpcA was found to be tolerant of cysteine site-directed mutagenesis, demonstrated by the successful expression of natively folded proteins bearing cysteine mutations at a series of sites selected to vary characteristically with respect to the three -CXXCH- heme binding domains. The introduced cysteines readily reacted with Ru(II)-(2,2'-bpy)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form covalently linked constructs that support both photo-oxidative and photo-reductive quenching of the photosensitizer excited state, depending upon the initial heme redox state. Excited-state electron-transfer times were found to vary from 6 × 10(-12) to 4 × 10(-8) s, correlated with the distance and pathways for electron transfer. The fastest rate is more than 10(3)-fold faster than previously reported for photosensitizer-redox protein constructs using amino acid residue linking. Clear evidence for inter-heme electron transfer within the multi-heme protein is not detected within the lifetimes of the charge-separated states. These results demonstrate an opportunity to develop multi-heme c-cytochromes for investigation of electron transfer in protein "molecular wires" and to serve as frameworks for metalloprotein designs that support multiple-electron-transfer redox chemistry.

Brownian-dynamics simulations of protein–protein interactions in the photosynthetic electron transport chain

The application of Brownian dynamics for simulation of transient protein-protein interactions is reviewed. The review focuses on theoretical basics of Brownian dynamics method, its particular implementations, advantages and drawbacks of the method. The outlook for future development of Brownian dynamics-based simulation techniques is discussed. Special attention is given to analysis of Brownian dynamics trajectories. The second part of the review is dedicated to the role of Brownian dynamics simulations in studying photosynthetic electron transport. Interactions of mobile electron carriers (plastocyanin, cytochrome c6, and ferredoxin) with their reaction partners (cytochrome b6f complex, photosystem I, ferredoxin:NADP-reductase, and hydrogenase) are considered.

Copper economy in Chlamydomonas: Prioritized allocation and reallocation of copper to respiration vs. photosynthesis

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

Analysis of twin-arginine translocation system gene homology and transcription in Vibrio species

To determine the function of twin-arginine translocation system (Tat) and gene cluster in Vibrio strains and to analyze the homology of tat gene cluster among different Vibrio spp. strains based on N16961 and tatABC mutant strains N169-dtat. Different serotypes of biotype strains of Vibrio spp. were selected to detect the transcription of 4 genes of Tat transport system and upstream ubi aarF gene and downstream cyt551 gene by the total RNA reverse transcription and homologicity of the gene cluster by sequencing analysis. Our results showed that the 4 genes of tat cluster (tatA, tatB, tatC, and tatE) were intragenic and co-transcribed. We found that ubi aarF gene could be co-transcribed with tatA, tatB, but not with tatC. The electron transport chain and energy metabolism-related genes, cytochrome C551 peroxidase gene, and 4 genes located at upstream of tatABC operon were not transcribed with tatABC. Although the co-transcription between ubi aarF and tatAB was blocked in N169-dtat strain, they were still transcribed separately. Homologous analysis of genes of tat cluster in different types of Vibrio cholerae showed that tat gene cluster was a very conservative. The ubi and aarF gene might be co-transcribed with genes of tat cluster in Vibrio cholerae, which and the close relationship showed that they might play a key function in Vibrio cholerae.

Effects of protein structure on iron-polypeptide vibrational dynamic coupling in cytochrome c.

Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm–1 region is dominated by NHis–Fe–SMet axial ligand and polypeptide motions, while the 325–400 cm–1 region shows dominant contributions from ν(Fe–NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed.

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

Cytochrome c555 from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c555 ) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 310 -α-310 helix containing the heme-ligating Met61. Herein, we show that AA cyt c555 forms dimers by swapping the region containing the extra 310 -α-310 helix and C-terminal α-helix. The asymmetric unit of the crystal of dimeric AA cyt c555 contained two dimer structures, where the structure of the hinge region (Val53-Lys57) was different among all four protomers. Dimeric AA cyt c555 dissociated to monomers at 92 ± 1°C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c555 were maintained at pH 2.2-11.0. CN(-) and CO bound to dimeric AA cyt c555 in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN(-) and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2-11.0), respectively, in a wide range of temperature (25-85°C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c555 changes upon dimerization by domain swapping.

Oligomerization enhancement and two domain swapping mode detection for thermostable cytochrome c552 via the elongation of the major hinge loop.

High-order oligomers of Hydrogenobacter thermophilus cytochrome c552 increased with the insertion of more Gly residues between Ala18 and Lys19 at the major hinge loop of the wild-type protein. N-Terminal domain swapping and C-terminal domain swapping were elucidated by using X-ray crystallography for the mutant with the insertion of three Gly residues at the hinge loop.

New Perspectives on Quinol Binding Motifs at the bc1 Complex Based on MD Simulations

The bc1 complex is a central player in the conversion of energy into ATP synthesis in photosynthesis and respiration, and its overall mechanism, the Q-cycle, is well known. However, the quinol-protein interaction that initiates Q-cycle at the Qo-binding site have not yet been described. Employing classical MD simulations in tandem with DFT calculations, the quinol binding motifs to the Qo-site of bc1 complex is investigated for a range of Qo-site protonation states. The computations revealed a novel configuration of the key side groups at the Qo-site site, such as H156, Y147 and E295, that stabilize the reaction complex and provide an optimal configuration prior to the charge transfer reactions between quinol and iron-sulfur cluster of the Rieske protein. Re-arrangements in the E295 and Y147 side chains were observed in all our simulations, showing intermediate bridging hydrogen bonding between quinol and E295, not observed before. Simulations were extended to study cytochrome c2 docking to cytochrome c1, required for the removal of electrons from the bc1 complex. We found, the docking interface is characterized by a semicircular arrangement of electrostatic residues that draws the cytochrome c2 and bc1 complex into an encounter complex, a hydrophobic minimal core to facilitate electron transfer, and mobility mismatch between the bound surfaces to induce post-electron transfer undocking.

Multimerization of Solution-State Proteins by Anionic Porphyrins

Protein-protein interactions and formation of multi-subunit protein complexes remain among the most challenging areas of modern Biophysics. Recently, surface binding and interactions of anionic porphyins with cationic proteins gained a lot of attention as relevant models for protein surface molecular recognition and photoinitiated electron transfer. However, interpretation of data in nearly all reports explicitly or implicitly assumed interaction of porphyrin with monodisperse proteins in solutions. Here, using small-angle X-ray scattering with solution phase samples, we demonstrate that horse heart cytochrome (cyt) c, triheme cytochrome c7 PpcA from Geobacter sulfurreducens, and hen egg lysozyme multimerize in the presence of several water-soluble porphyrins. Multimerization of cyt c induced by tetrakis(4-sulfonatophenyl)porphyrin showed a pH dependence with a stronger apparent binding affinity under alkaline conditions and was weakened in the presence of a high salt concentration. Ferric-cyt c formed complexes larger than those formed by ferro-cyt c. Free base TPPS and FeTPPS facilitated formation of complexes larger than those of ZnTPPS. A number of carboxylated porphyrins induced multimer formation as well. No increase in protein aggregation state for cationic proteins was observed in the presence of cationic porphyrins or sulfonated anthraquinone. All-atom molecular dynamics simulations of cyt c and PpcA with free base TPPS corroborated X-ray scattering results and revealed a mechanism by which the tetrasubstituted charged porphyrins serve as bridging ligands nucleating multimerization of the complementarily charged protein. The final aggregation products suggest that multimerization involves a combination of electrostatic and hydrophobic interactions. The results demonstrate an overlooked complexity in the design of multifunctional ligands for protein surface recognition.

Cytochrome c-based domain modularity governs genus-level diversification of electron transfer to dissimilatory nitrite reduction.

The genus Neisseria contains two pathogenic species (N. meningitidis and N. gonorrhoeae) in addition to a number of commensal species that primarily colonize mucosal surfaces in man. Within the genus, there is considerable diversity and apparent redundancy in the components involved in respiration. Here, we identify a unique c-type cytochrome (cN ) that is broadly distributed among commensal Neisseria, but absent in the pathogenic species. Specifically, cN supports nitrite reduction in N. gonorrhoeae strains lacking the cytochromes c5 and CcoP established to be critical to NirK nitrite reductase activity. The c-type cytochrome domain of cN shares high sequence identity with those localized c-terminally in c5 and CcoP and all three domains were shown to donate electrons directly to NirK. Thus, we identify three distinct but paralogous proteins that donate electrons to NirK. We also demonstrate functionality for a N. weaverii NirK variant with a C-terminal c-type heme extension. Taken together, modular domain distribution and gene rearrangement events related to these respiratory electron carriers within Neisseria are concordant with major transitions in the macroevolutionary history of the genus. This work emphasizes the importance of denitrification as a selectable trait that may influence speciation and adaptive diversification within this largely host-restricted bacterial genus.

Global transcriptional profiles of the copper responses in the cyanobacterium Synechocystis sp. PCC 6803.

Copper is an essential element involved in fundamental processes like respiration and photosynthesis. However, it becomes toxic at high concentration, which has forced organisms to control its cellular concentration. We have recently described a copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803, which is mediated by the two-component system, CopRS, a RND metal transport system, CopBAC and a protein of unknown function, CopM. Here, we report the transcriptional responses to copper additions at non-toxic (0.3 µM) and toxic concentrations (3 µM) in the wild type and in the copper sensitive copR mutant strain. While 0.3 µM copper slightly stimulated metabolism and promoted the exchange between cytochrome c6 and plastocyanin as soluble electron carriers, the addition of 3 µM copper catalyzed the formation of ROS, led to a general stress response and induced expression of Fe-S cluster biogenesis genes. According to this, a double mutant strain copRsufR, which expresses constitutively the sufBCDS operon, tolerated higher copper concentration than the copR mutant strain, suggesting that Fe-S clusters are direct targets of copper toxicity in Synechocystis. In addition we have also demonstrated that InrS, a nickel binding transcriptional repressor that belong to the CsoR family of transcriptional factor, was involved in heavy metal homeostasis, including copper, in Synechocystis. Finally, global gene expression analysis of the copR mutant strain suggested that CopRS only controls the expression of copMRS and copBAC operons in response to copper.

All-atom molecular dynamics simulation of a photosystem i/detergent complex.

All-atom molecular dynamics (MD) simulation was used to investigate the solution structure and dynamics of the photosynthetic pigment-protein complex photosystem I (PSI) from Thermosynechococcus elongatus embedded in a toroidal belt of n-dodecyl-β-d-maltoside (DDM) detergent. Evaluation of root-mean-square deviations (RMSDs) relative to the known crystal structure show that the protein complex surrounded by DDM molecules is stable during the 200 ns simulation time, and root-mean-square fluctuation (RMSF) analysis indicates that regions of high local mobility correspond to solvent-exposed regions such as turns in the transmembrane α-helices and flexible loops on the stromal and lumenal faces. Comparing the protein-detergent complex to a pure detergent micelle, the detergent surrounding the PSI trimer is found to be less densely packed but with more ordered detergent tails, contrary to what is seen in most lipid bilayer models. We also investigated any functional implications for the observed conformational dynamics and protein-detergent interactions, discovering interesting structural changes in the psaL subunits associated with maintaining the trimeric structure of the protein. Importantly, we find that the docking of soluble electron mediators such as cytochrome c6 and ferredoxin to PSI is not significantly impacted by the solubilization of PSI in detergent.

Electrochemical Behavior of Cytochrome c552 from a Psychrophilic Microorganism

Psychrophilic organisms play a significant role in global nutrient cycling and bioremediation, and thus there is great interest in understanding the details of their cellular processes. Here, we report the first electrochemical measurements of cytochrome c552 isolated from the model psychrophile, Colwellia psychrerythraea. Quasi-reversible redox behavior is observed at thiol-modified and bare gold electrodes with midpoint potentials of 247 and 277 mV vs SHE, respectively. Comparison of the voltammetric response on the two substrates illustrates the need for a modified surface to direct protein orientation and facilitate electron transfer in a reproducible manner. Temperature-dependence measurements of the current profile illustrate the protein’s ability to participate in electron transfer reactions over a 50° range, which is supported by recent structural evidence of enhanced flexibility. These electrochemical findings allow the behavior of this psychrophilic cytochrome to be evaluated in the context of m...

Multimerization of solution-state proteins by tetrakis(4-sulfonatophenyl)porphyrin.

Surface binding and interactions of anionic porphyins bound to cationic proteins have been studied for nearly three decades and are relevant as models for protein surface molecular recognition and photoinitiated electron transfer. However, interpretation of data in nearly all reports explicitly or implicitly assumed interaction of porphyrin with monodisperse proteins in solutions. In this report, using small-angle X-ray scattering with solution phase samples, we demonstrate that horse heart cytochrome (cyt) c, triheme cytochrome c7 PpcA from Geobacter sulfurreducens, and hen egg lysozyme multimerize in the presence of zinc tetrakis(4-sulfonatophenyl)porphyrin (ZnTPPS). Multimerization of cyt c showed a pH dependence with a stronger apparent binding affinity under alkaline conditions and was weakened in the presence of a high salt concentration. Ferric-cyt c formed complexes larger than those formed by ferro-cyt c. Free base TPPS and FeTPPS facilitated formation of complexes larger than those of ZnTPPS. No increase in protein aggregation state for cationic proteins was observed in the presence of cationic porphyrins. All-atom molecular dynamics simulations of cyt c and PpcA with free base TPPS corroborated X-ray scattering results and revealed a mechanism by which the tetrasubstituted charged porphyrins serve as bridging ligands nucleating multimerization of the complementarily charged protein. The final aggregation products suggest that multimerization involves a combination of electrostatic and hydrophobic interactions. The results demonstrate an overlooked complexity in the design of multifunctional ligands for protein surface recognition.

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase.

Working with “H2S”: Facts and apparent artifacts

Hydrogen sulfide (H2S) is an important signaling molecule with physiological endpoints similar to those of nitric oxide (NO). Growing interest in its physiological roles and pharmacological potential has led to large sets of contradictory data. The principle cause of these discrepancies can be the common neglect of some of the basic H2S chemistry. This study investigates how the experimental outcome when working with H2S depends on its source and dose and the methodology employed. We show that commercially available NaHS should be avoided and that traces of metal ions should be removed because these can reduce intramolecular disulfides and change protein structure. Furthermore, high H2S concentrations may lead to a complete inhibition of cell respiration, mitochondrial membrane potential depolarization and superoxide generation, which should be considered when discussing the biological effects observed upon treatment with high concentrations of H2S. In addition, we provide chemical evidence that H2S can directly react with superoxide. H2S is also capable of reducing cytochrome c3+ with the concomitant formation of superoxide. H2S does not directly react with nitrite but with NO electrodes that detect H2S. In addition, H2S interferes with the Griess reaction and should therefore be removed from the solution by Cd2+ or Zn2+ precipitation prior to nitrite quantification. 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) is reduced by H2S, and its use should be avoided in combination with H2S. All these constraints must be taken into account when working with H2S to ensure valid data.

Molecular organization of cytochrome c2 near the binding domain of cytochrome bc1 studied by electron spin-lattice relaxation enhancement.

Measurements of specific interactions between proteins are challenging. In redox systems, interactions involve surfaces near the attachment sites of cofactors engaged in interprotein electron transfer (ET). Here we analyzed binding of cytochrome c2 to cytochrome bc1 by measuring paramagnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c2. PRE was exclusively induced by the iron atom of heme c1 of cytochrome bc1, which guaranteed that only the configurations with SL to heme c1 distances up to ∼30 Å were detected. Changes in PRE were used to qualitatively and quantitatively characterize the binding. Our data suggest that at low ionic strength and under an excess of cytochrome c2 over cytochrome bc1, several cytochrome c2 molecules gather near the binding domain forming a “cloud” of molecules. When the cytochrome bc1 concentration increases, the cloud disperses to populate additional available binding domains. An increase in ionic strength weakens the attractive forces and the average distance between cytochrome c2 and cytochrome bc1 increases. The spatial arrangement of the protein complex at various ionic strengths is different. Above 150 mM NaCl the lifetime of the complexes becomes so short that they are undetectable. All together the results indicate that cytochrome c2 molecules, over the range of salt concentration encompassing physiological ionic strength, do not form stable, long-lived complexes but rather constantly collide with the surface of cytochrome bc1 and ET takes place coincidentally with one of these collisions.

Azurin–DNA Conjugate with the Binding Motif of a Transcriptional Regulator, CooA: CO-dependent Modulation of the Electron-transfer Reaction

Abstract An azurin–DNA conjugate (Azu–DNA) where the DNA part contains the binding sequence of a carbon monoxide (CO)-dependent transcriptional regulator, CooA, has been prepared. CooA in the CO-bound form (CO–CooA) showed specific binding to Azu–DNA. The association of CO–CooA with Azu–DNA affects the electron transfer (ET) between Azu–DNA and its redox partner protein, cytochrome c552. Consequently, the CO-dependent modulation of ET was achieved. This could be a potential platform for a novel biobased sensor, which exploits the transcriptional regulator as a detection module and whose readout is immediately transduced to a readily processable electronic signal.