Cytochromes C Research Articles

Properties and structure of a low-potential, penta-heme cytochrome c552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum.

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c555.

Highly-ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c555 , and assembled BBP into a triangle-shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain-swapping hinge loop of cyt c555 and connecting the original N- and C-terminal α-helices with an α-helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X-ray crystallographic analysis of the BBP trimer, the N-terminal region of one BBP molecule interacted intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N- and C-terminal α-helices with an α-helical linker may be useful for constructing organized protein structures.

The effect of cycA overexpression on hydrogen production performance of Rhodobacter sphaeroides HY01

The gene cycA encodes a periplasmic protein, cytochrome c2 (cyt c2), which dominates electron transfer from the membrane-bound ubiquinol: cyt c2 oxidoreductase (cyt bc1) to the photosynthetic reaction center, contributing to the production of transmembrane proton potential and then the synthesis of ATP. For photosynthetic bacteria, the total energy supply for light anaerobic growth and hydrogen production comes from photophosphorylation. As a result, the key protein encoding gene plays an important role in hydrogen production. To figure out the specific effect of cycA expression level on H2 production ability of Rhodobacter sphaeroides HY01, cycA-expression plasmids derived from pRK415 and pBBR1MCS-2 were constructed and then crossed into the parent strain R. sphaeroides HY01 for H2 production test. And further verification by RT-PCR suggested that there was about 20% enhancement of cycA expression level by pBBR1MCS-2 where the H2 production performance of corresponding strain was improved by 6–8% compared with blank control. In contrast, cycA expression level was about 3.4 folds by vector pRK415 compared with control strain, but corresponding strains showed slightly depressed H2 production performance. Besides, the mutant XJ01 with cycA gene overexpressing by 70% in the genome of HY01(hupSL:cycA) also showed positive effect on hydrogen production performance. The results demonstrated that slightly overexpression of cycA could enhance the hydrogen production rate, but too much higher level of cycA-expression could show negative effect on H2 production performance of R. sphaeroides HY01.

Identification and Analysis of a Novel Gene Cluster Involves in Fe2+ Oxidation in Acidithiobacillus ferrooxidans ATCC 23270, a Typical Biomining Acidophile.

Iron-oxidizing Acidithiobacillus spp. are applied worldwide in biomining industry to extract metals from sulfide minerals. They derive energy for survival through Fe2+ oxidation and generate Fe3+ for the dissolution of sulfide minerals. However, molecular mechanisms of their iron oxidation still remain elusive. A novel two-cytochrome-encoding gene cluster (named tce gene cluster) encoding a high-molecular-weight cytochrome c (AFE_1428) and a c4-type cytochrome c552 (AFE_1429) in A. ferrooxidans ATCC 23270 was first identified in this study. Bioinformatic analysis together with transcriptional study showed that AFE_1428 and AFE_1429 were the corresponding paralog of Cyc2 (AFE_3153) and Cyc1 (AFE_3152) which were encoded by the extensively studied rus operon and had been proven involving in ferrous iron oxidation. Both AFE_1428 and AFE_1429 contained signal peptide and the classic heme-binding motif(s) as their corresponding paralog. The modeled structure of AFE_1429 showed high resemblance to Cyc1. AFE_1428 and AFE_1429 were preferentially transcribed as their corresponding paralogs in the presence of ferrous iron as sole energy source as compared with sulfur. The tce gene cluster is highly conserved in the genomes of four phylogenetic-related A. ferrooxidans strains that were originally isolated from different sites separated with huge geographical distance, which further implies the importance of this gene cluster. Collectively, AFE_1428 and AFE_1429 involve in Fe2+ oxidation like their corresponding paralog by integrating with the metalloproteins encoded by rus operon. This study provides novel insights into the Fe2+ oxidation mechanism in Fe2+-oxidizing A. ferrooxidans ssp.

The Stability, Reduction Potential and Ligand State of Two Conformations of a C-Type Cytochrome from the Diatom Thalassiosira Pseudonana

In this study we investigate the structure and function of a c-type cytochrome from the diatom Thalassiosira pseudonana, Tp34211. The axial iron ligand of a c-type cytochrome is often either a methionine or histidine residue. The axial ligand plays an important role in determining the midpoint potential of the heme cofactor, which in turn determines the possible physiological electron donors and acceptors. Initial experiments suggested that recombinantly expressed Tp34211 forms two alternative conformations that can be differentiated by Soret band changes upon imidazole binding or quenching of the tryptophan fluorescence emission signal. Based on sequence alignments of Tp34211 and known cytochrome c6 proteins, we predict that Tp34211 has an axial histidine ligand (H78), but a neighboring lysine residue (K79) could serve as an alternate ligand. To test the possible role of ligand switching in the two alternative conformations observed for Tp34211, we recombinantly expressed and purified both wild-type Tp34211 and a K79A variant that should force bis-His ligation. Following purification by ion-exchange and size-exclusion chromatogrpahy, guanidine induced unfolding assays were performed on the various protein samples, with extent of unfolding measured via intrinsic tryptophan fluorescence. The ΔG°unfolding of the unquenched conformation of WT Tp34211 and the K79A mutant were determined to be the same within error, suggesting that histidine coordination is favored under normal solution conditions. Midpoint potentials of the K79A variant and the two conformations of the wild-type protein will also be measured to further identify the role of histidine versus lysine ligation in Tp34211.

Comparison of the Stability and Reduction Potential of Cytochrome C6 Proteins from a Psychrophilic and a Mesophilic Diatom

Comparison of the Stability and Reduction Potential of Cytochrome C6 Proteins from a Psychrophilic and a Mesophilic Diatom

Electromagnetic Radiation Disturbed the Photosynthesis of Microcystis aeruginosa at the Proteomics Level

Photosynthesis of Microcystis aeruginosa under Electromagnetic Radiation (1.8 GHz, 40 V/m) was studied by using the proteomics. A total of 30 differentially expressed proteins, including 15 up-regulated and 15 down-regulated proteins, were obtained in this study. The differentially expressed proteins were significantly enriched in the photosynthesis pathway, in which the protein expression levels of photosystems II cytochrome b559 α subunit, cytochrome C550, PsbY, and F-type ATP synthase (a, b) decreased. Our results indicated that electromagnetic radiation altered the photosynthesis-related protein expression levels, and aimed at the function of photosynthetic pigments, photosystems II potential activity, photosynthetic electron transport process, and photosynthetic phosphorylation process of M. aeruginosa. Based on the above evidence, that photoreaction system may be deduced as a target of electromagnetic radiation on the photosynthesis in cyanobacteria; the photoreaction system of cyanobacteria is a hypothetical “shared target effector” that responds to light and electromagnetic radiation; moreover, electromagnetic radiation does not act on the functional proteins themselves but their expression processes.

Photooxidation and photoreduction of exogenous cytochrome c by photosystem II preparations after various modifications of the water-oxidizing complex

The redox interaction of exogenous cytochrome c550 (Cyt) with PSII isolated from spinach was studied. Illumination of PSII particles in the presence of Cyt led to: (1) Cyt photooxidation by PSII reaction center (demonstrated at the first time), (2) Cyt photoreduction via O2−• photoproduced on the acceptor side of PSII, and (3) Cyt photoreduction by reduced electron carriers of PSII. A step-by-step removal of components of water-oxidizing complex was accompanied by the appearance of Cyt photooxidation, an increase in the superoxide dismutase (SOD)-dependent Cyt photoreduction (related to O2−• formation), and a decrease in the SOD-independent Cyt photoreduction. Re-addition of PsbO protein diminished the Cyt-induced restoration of electron transfer in PSII. Addition of diuron led to inhibition of these photoprocesses, while exogenous Mn2+ inhibited only the Cyt c photooxidation. The results can be important for correct measurements of O2−• photoproduction in PSII and for elucidation of the role of cytochrome c550 in cyanobacterial PSII.

Electron Paramagnetic Resonance Characterization of the Triheme Cytochrome from Geobacter sulfurreducens.

Periplasmic cytochrome A (PpcA) is a representative of a broad class of multiheme cytochromes functioning as protein "nanowires" for storage and extracellular transfer of multiple electrons in the δ-proteobacterium Geobacter sulfurreducens. PpcA contains three bis-His coordinated hemes held in a spatial arrangement that is highly conserved among the multiheme cytochromes c3 and c7 families, carries low potential hemes, and is notable for having one of the lowest number of amino acids utilized to maintain a characteristic protein fold and site-specific heme function. Low temperature X-band electron paramagnetic resonance (EPR) spectroscopy has been used to characterize the electronic configuration of the Fe(III) and the ligation mode for each heme. The three sets of EPR signals are assigned to individual hemes in the three-dimensional crystal structure. The relative energy levels of the Fe(III) 3d orbitals for individual hemes were estimated from the principal g-values. The observed g-tensor anisotropy was used as a probe of electronic structure of each heme, and differences were determined by specifics of axial ligation. To ensure unambiguous assignment of highly anisotropic low-spin (HALS) signal to individual hemes, EPR analyses of iron atom electronic configurations have been supplemented with investigation of porphyrin macrocycles by one-dimensional 1H NMR chemical shift patterns for the methyl substituents. Within optimized geometry of hemes in PpcA, the magnetic interactions between hemes were found to be minimal, similar to the c3 family of tetraheme cytochromes.

Requirements for Efficient Thiosulfate Oxidation in Bradyrhizobium diazoefficiens.

One of the many disparate lifestyles of Bradyrhizobium diazoefficiens is chemolithotrophic growth with thiosulfate as an electron donor for respiration. The employed carbon source may be CO2 (autotrophy) or an organic compound such as succinate (mixotrophy). Here, we discovered three new facets of this capacity: (i) When thiosulfate and succinate were consumed concomitantly in conditions of mixotrophy, even a high molar excess of succinate did not exert efficient catabolite repression over the use of thiosulfate. (ii) Using appropriate cytochrome mutants, we found that electrons derived from thiosulfate during chemolithoautotrophic growth are preferentially channeled via cytochrome c550 to the aa3-type heme-copper cytochrome oxidase. (iii) Three genetic regulators were identified to act at least partially in the expression control of genes for chemolithoautotrophic thiosulfate oxidation: RegR and CbbR as activators, and SoxR as a repressor.

Fluorescence induction of photosynthetic bacteria

The kinetics of bacteriochlorophyll fluorescence in intact cells of the purple nonsulfur bacterium Rhodobacter sphaeroides were measured under continuous and pulsed actinic laser diode (808 nm wavelength and maximum 2 W light power) illumination on the micro- and millisecond timescale. The fluorescence induction curve was interpreted in terms of a combination of photochemical and triplet fluorescence quenchers and was demonstrated to be a reflection of redox changes and electron carrier dynamics. By adjustment of the conditions of single and multiple turnovers of the reaction center, we obtained 11 ms–1 and 120 μs–1 for the rate constants of cytochrome c23+ detachment and cyclic electron flow, respectively. The effects of cytochrome c2 deletion and chemical treatments of the bacteria and the advantages of the fluorescence induction study on the operation of the electron transport chain in vivo were discussed.

Insights into the recognition and electron transfer steps in nitric oxide reductase from Marinobacter hydrocarbonoclasticus

Marinobacter hydrocarbonoclasticus nitric oxide reductase, cNOR, is an integral membrane protein composed of two subunits with different roles, NorC (electron transfer) and NorB (catalytic) that receives electrons from the soluble cytochrome c552 and reduces nitric oxide to nitrous oxide in the denitrification pathway. The solvent-exposed domain of NorC, harboring a c-type heme was heterologously produced, along with its physiological electron donor, cytochrome c552. These two proteins were spectroscopically characterized and shown to be similar to the native proteins, both being low-spin and Met-His coordinated, with the soluble domain of NorC presenting some additional features of a high-spin heme, which is consistent with the higher solvent accessibility of its heme and weaker coordination of the methionine axial ligand. The electron transfer complex between the two proteins has a 1:1 stoichiometry, and an upper limit for the dissociation constant was estimated by 1H NMR titration to be 1.2±0.4μM. Electrochemical techniques were used to characterize the interaction between the proteins, and a model structure of the complex was obtained by molecular docking. The electrochemical observations point to the modulation of the NorC reduction potential by the presence of NorB, tuning its ability to receive electrons from cytochrome c552.

Density functional study of porphyrin distortion effects on redox potential of heme.

Heme is involved in various biochemical roles in hemoproteins. In the present study, the effect of heme distortion on the redox potential was systematically investigated with density functional calculations. We focused on the ruffled and saddled distortions of heme, which correspond to the two lowest-frequency normal modes. Our computations demonstrated that the ruffled distortion tended to reduce the redox potential of heme and that the transition of the electronic configuration occurred from (dxz , dyz )3 (dxy )2 to (dxz , dyz )4 (dxy )1 . In contrast, the saddled distortion had a tendency toward an increase in the redox potential, and no transition of the electronic configuration occurred. In experiments, these tendencies were found in the relationship between with the heme distortions and the redox potentials in cytochrome c3 . © 2017 Wiley Periodicals, Inc.

Total electron acceptor loading and composition affect hexavalent uranium reduction and microbial community structure in a membrane biofilm reactor

Molecular microbiology tools (i.e., 16S rDNA gene sequencing) were employed to elucidate changes in the microbial community structure according to the total electron acceptor loading (controlled by influent flow rate and/or medium composition) in a H2-based membrane biofilm reactor evaluated for removal of hexavalent uranium. Once nitrate, sulfate, and dissolved oxygen were replaced by U(VI) and bicarbonate and the total acceptor loading was lowered, slow-growing bacteria capable of reducing U(VI) to U(IV) dominated in the biofilm community: Replacing denitrifying bacteria Rhodocyclales and Burkholderiales were spore-producing Clostridiales and Natranaerobiales. Though potentially competing for electrons with U(VI) reducers, homo-acetogens helped attain steady U(VI) reduction, while methanogenesis inhibited U(VI) reduction. U(VI) reduction was reinstated through suppression of methanogenesis by addition of bromoethanesulfonate or by competition from SRB when sulfate was re-introduced. Predictive metagenome analysis further points out community changes in response to alterations in the electron-acceptor loading: Sporulation and homo-acetogenesis were critical factors for strengthening stable microbial U(VI) reduction. This study documents that sporulation was important to long-term U(VI) reduction, whether or not microorganisms that carry out U(VI) reduction mediated by cytochrome c3, such as SRB and ferric-iron-reducers, were inhibited.

Maturation of Plastid c-type Cytochromes.

Cytochromes c are hemoproteins, with the prosthetic group covalently linked to the apoprotein, which function as electron carriers. A class of cytochromes c is defined by a CXXCH heme-binding motif where the cysteines form thioether bonds with the vinyl groups of heme. Plastids are known to contain up to three cytochromes c. The membrane-bound cytochrome f and soluble cytochrome c6 operate in photosynthesis while the activity of soluble cytochrome c6A remains unknown. Conversion of apo- to holocytochrome c occurs in the thylakoid lumen and requires the independent transport of apocytochrome and heme across the thylakoid membrane followed by the stereospecific attachment of ferroheme via thioether linkages. Attachment of heme to apoforms of plastid cytochromes c is dependent upon the products of the CCS (for cytochrome c synthesis) genes, first uncovered via genetic analysis of photosynthetic deficient mutants in the green alga Chlamydomonas reinhardtii. The CCS pathway also occurs in cyanobacteria and several bacteria. CcsA and CCS1, the signature components of the CCS pathway are polytopic membrane proteins proposed to operate in the delivery of heme from the stroma to the lumen, and also in the catalysis of the heme ligation reaction. CCDA, CCS4, and CCS5 are components of trans-thylakoid pathways that deliver reducing equivalents in order to maintain the heme-binding cysteines in a reduced form prior to thioether bond formation. While only four CCS components are needed in bacteria, at least eight components are required for plastid cytochrome c assembly, suggesting the biochemistry of thioether formation is more nuanced in the plastid system.

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length.

Reversible denaturation of Pseudomonas aeruginosa cytochrome c551 (PAc551) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc551 denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc551 denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc551, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.

Electrochemical study of an electron shuttle diheme protein: The cytochrome c550 from T. thermophilus

Cytochrome c550, a diheme protein from the thermophilic bacterium Thermus thermophilus, is involved in an alternative respiration pathway allowing the detoxification of sulfite ions. It transfers the two electrons released from the oxidation of sulfite in a sulfite:cytochrome c oxidoreductase (SOR) enzyme to heme/copper oxidases via the monoheme cytochrome c552. It consists of two conformationally independent and structurally different domains (the C- and N-terminal) connected by a flexible linker. Both domains harbor one heme moiety. We report here the redox properties of the full-length protein and the individual C- and N-terminal fragments. We show by UV/Vis and EPR potentiometric titrations that the two fragments exhibit very similar potentials, despite their different environments. In the full-length protein, however, the N-terminal heme is easier to reduce than the C-terminal one, due to cooperative interactions. This finding is consistent with the kinetic measurements which showed that the N-terminal domain only accepts electrons from the SOR. Cytochrome c552 is able to interact with its partners both through electrostatic and hydrophobic interactions as could be shown by measuring efficient electron transfer at gold electrodes modified with charged and hydrophobic groups, respectively. The coupling of electrochemistry with infrared spectroscopy allowed us to monitor the conformational changes induced by electron transfer to each heme separately and to both simultaneously.

Chemical property based sequence characterization of PpcA and its homolog proteins PpcB-E: A mathematical approach.

Periplasmic c7 type cytochrome A (PpcA) protein is determined in Geobacter sulfurreducens along with its other four homologs (PpcB-E). From the crystal structure viewpoint the observation emerges that PpcA protein can bind with Deoxycholate (DXCA), while its other homologs do not. But it is yet to be established with certainty the reason behind this from primary protein sequence information. This study is primarily based on primary protein sequence analysis through the chemical basis of embedded amino acids. Firstly, we look for the chemical group specific score of amino acids. Along with this, we have developed a new methodology for the phylogenetic analysis based on chemical group dissimilarities of amino acids. This new methodology is applied to the cytochrome c7 family members and pinpoint how a particular sequence is differing with others. Secondly, we build a graph theoretic model on using amino acid sequences which is also applied to the cytochrome c7 family members and some unique characteristics and their domains are highlighted. Thirdly, we search for unique patterns as subsequences which are common among the group or specific individual member. In all the cases, we are able to show some distinct features of PpcA that emerges PpcA as an outstanding protein compared to its other homologs, resulting towards its binding with deoxycholate. Similarly, some notable features for the structurally dissimilar protein PpcD compared to the other homologs are also brought out. Further, the five members of cytochrome family being homolog proteins, they must have some common significant features which are also enumerated in this study.

Electrostatic roles in electron transfer from [NiFe] hydrogenase to cytochrome c3 from Desulfovibrio vulgaris Miyazaki F

Electrostatic interactions between proteins are key factors that govern the association and reaction rate. We spectroscopically determine the second-order reaction rate constant (k) of electron transfer from [NiFe] hydrogenase (H2ase) to cytochrome (cyt) c3 at various ionic strengths (I). The k value decreases with I. To analyze the results, we develop a semi-analytical formula for I dependence of k based on the assumptions that molecules are spherical and the reaction proceeds via a transition state. Fitting of the formula to the experimental data reveals that the interaction occurs in limited regions with opposite charges and with radii much smaller than those estimated from crystal structures. This suggests that local charges in H2ase and cyt c3 play important roles in the reaction. Although the crystallographic data indicate a positive electrostatic potential over almost the entire surface of the proteins, there exists a small region with negative potential on H2ase at which the electron transfer from H2ase to cyt c3 may occur. This local negative potential region is identical to the hypothetical interaction sphere predicted by the analysis. Furthermore, I dependence of k is predicted by the Adaptive Poisson–Boltzmann Solver considering all charges of the amino acids in the proteins and the configuration of H2ase/cyt c3 complex. The calculation reproduces the experimental results except at extremely low I. These results indicate that the stabilization derived from the local electrostatic interaction in the H2ase/cyt c3 complex overcomes the destabilization derived from the electrostatic repulsion of the overall positive charge of both proteins.

Biological Semiconductors: Structural Control of Heme Redox Potentials in PpcA, a 3-Heme Cytochrome

In an effort to tune and optimize relative bandgap potentials found in biological nanowires that form from multiheme domains of members of the cytochrome c7 family, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were successfully expressed in E.coli and show thermal stability in temperature-dependent CD experiments comparable to the wild-type protein (Tm > 90°C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins formed the expected compact globular structures. In addition to ongoing attempts to obtain protein crystals for X-ray crystallography, we performed extensive all-atom molecular dynamics simulations for all mutants. Formation of propionate hydrogen bonds is supported by simulations for A19R and A23R, while increased solvent access is supported for A19I, A19R, A23R, H20M, and K60E. UV/Vis and NMR redox titrations were performed in order to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties.