Cyt B562 Research Articles

Computationally Guided Redesign of a Heme-free Cytochrome with Native-like Structure and Stability.

Metals can play key roles in stabilizing protein structures, but ensuring their proper incorporation is a challenge when a metalloprotein is overexpressed in a non-native cellular environment. Here, we have used computational protein design tools to redesign cytochrome b562 (cyt b562), which relies on the binding of its heme cofactor to achieve its proper fold, into a stable, heme-free protein. The resulting protein, ApoCyt, features only four mutations and no metal-ligand or covalent bonds, yet displays improved stability over cyt b562. Mutagenesis studies and X-ray crystal structures reveal that the increase in stability is due to the computationally prescribed mutations, which stabilize the protein fold through a combination of hydrophobic packing interactions, hydrogen bonds, and cation-π interactions. Upon installation of the relevant mutations, ApoCyt is capable of assembling into previously reported, cytochrome-based trimeric and tetrameric assemblies, demonstrating that ApoCyt retains the structure and assembly properties of cyt b562. The successful design of ApoCyt therefore enables further functional diversification of cytochrome-based assemblies and demonstrates that structural metal cofactors can be replaced by a small number of well-designed, non-covalent interactions.

Allergens of the urushiol family promote mitochondrial dysfunction by inhibiting the electron transport at the level of cytochromes b and chemically modify cytochrome c1

BackgroundUrushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response.ResultsElectron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562).ConclusionOur data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols.

Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b562 as an electron transfer domain

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Methane generation via intraprotein C–S bond cleavage in cytochrome b562 reconstituted with nickel didehydrocorrin

Cytochrome b562 (Cyt b562) reconstituted with nickel didehydrocorrin (NiII(DDHC)), a protein-based functional model of methyl-coenzyme M reductase (MCR), was investigated to demonstrate methane generation via intraprotein cleavage of a C–S bond. NiII(DDHC) was synthesized as a model complex of an MCR cofactor known as F430 and found to show NiII/NiI redox behavior with a potential of −0.61 V vs. Ag|AgCl. This potential is slightly positive-shifted compared to that of F430 without protein. Conjugation of NiII(DDHC) with the apo-form of Cyt b562 provides reconstituted Cyt b562 (rCyt b562(NiII(DDHC))) which was characterized by spectroscopic measurements. Photoirradiation of rCyt b562(NiII(DDHC)) generates methane gas in the presence of tris(2,2′-bipyridine)ruthenium(II) chloride as a photosensitizer and sodium ascorbate as a sacrificial reagent. Further experiments using Cyt b562 mutants indicate that methane is derived from the CH3S group of the methionine residue in the heme-binding site where thioether, thiol and the nickel center are precisely arranged. The present study demonstrates the first example of methane generation via intraprotein cleavage of a C–S bond using a functional model of MCR.

Photoinduced electron transfer within supramolecular hemoprotein co-assemblies and heterodimers containing Fe and Zn porphyrins

Electron transfer (ET) events occurring within metalloprotein complexes are among the most important classes of reactions in biological systems. This report describes a photoinduced electron transfer between Zn porphyrin and Fe porphyrin within a supramolecular cytochrome b562 (Cyt b562) co-assembly or heterodimer with a well-defined rigid structure formed by a metalloporphyrin–heme pocket interaction and a hydrogen-bond network at the protein interface. The photoinduced charge separation (CS: kCS = 320–600 s−1) and subsequent charge recombination (CR: kCR = 580–930 s−1) were observed in both the Cyt b562 co-assembly and the heterodimer. In contrast, interestingly, no ET events were observed in a system comprised of a flexible and structurally-undefined co-assembly and heterodimers which lack the key hydrogen-bond interaction at the protein interface. Moreover, analysis of the kinetic constants of CS and CR of the heterodimer using the Marcus equation suggests that a single-step ET reaction occurs in the system. These findings provide strong support that the rigid hemoprotein-assembling system containing an appropriate hydrogen-bond network at the protein interface is essential for monitoring the ET reaction.

Defined covalent assembly of protein molecules on graphene using a genetically encoded photochemical reaction handle.

We have created modified protein variants by introducing a non-canonical amino acid p-azido-l-phenylalanine (azF) into defined positions for photochemically-induced covalent attachment to graphene. Attachment of GFP, TEM and cyt b562 proteins was verified through a combination of atomic force and scanning tunnelling microscopy, resistance measurements, Raman data and fluorescence measurements. This method can in principle be extended to any protein which can be engineered in this way without adversely affecting its structural stability.

Reengineering cyt b562 for hydrogen production: A facile route to artificial hydrogenases

Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Domain-swapped cytochrome cb562 dimer and its nanocage encapsulating a Zn-SO4 cluster in the internal cavity.

Protein nanostructures have been gaining in interest, along with developments in new methods for construction of novel nanostructures. We have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt cb562, with the cyt b562 heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt cb562 did not dissociate to monomers at 4 °C, whereas dimeric cyt b562 dissociated under the same conditions, showing that heme attachment to the protein moiety stabilizes the domain-swapped structure. According to X-ray crystallographic analysis of dimeric cyt cb562, the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer, where Lys51-Asp54 served as a hinge loop. The heme coordination structure of the dimer was similar to that of the monomer. In the crystal, three domain-swapped cyt cb562 dimers formed a unique cage structure with a Zn-SO4 cluster inside the cavity. The Zn-SO4 cluster consisted of fifteen Zn2+ and seven SO42- ions, whereas six additional Zn2+ ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn2+ ions and connection of two four-helix bundle units through the conformation-adjustable hinge loop. These results show that domain swapping can be applied in the construction of unique protein nanostructures.

Reduction of Iron Center Enhances the Mechanical Stability of Cytochrome B562

Because redox energy can be converted into conformational energy, heme redox proteins offer a unique opportunity to examine the coupling between redox reactions and protein mechanics. Here, we use Atomic Force Microscopy-based single-molecule force spectroscopy (SMFS) to directly examine the effect of heme and its oxidation state on the mechanical properties of cytochrome b562 (cyt b562). We found that cyt b562 is mechanically stronger in its reduced state as compared to its oxidized state. In addition, we discovered the shortening of the folded length of cytochrome b562 in its reduced state. This novel observation could be the basis of heme protein-based “piezoelectric-like” nanomaterials and actuators.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Supramolecular Linear Assemblies of Cytochrome b 562 Immobilized on a Gold Electrode

Linear assemblies of cytochrome b562 (cyt b562) containing a chemically-modified heme molecule on the protein surface were constructed by successive intermolecular heme–heme pocket interactions and then immobilized onto a gold electrode via a heme molecule anchored on the electrode. The accumulation of the cyt b562 units were confirmed by electrochemical analyses. The average number of proteins in each assembly was estimated to be about 6 units by quartz crystal microbalance and atomic force microscopy (AFM) analyses. The linear assemblies of the cyt b562 units were clearly visualized by AFM as a rod-like architecture with a vertical orientation to the electrode surface.

Structural Basis for Efficient Chromophore Communication and Energy Transfer in a Constructed Didomain Protein Scaffold

The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.

Interfacial Construction of Gold Nanoshells on 3-Aminopropyl Triethoxysilane Modified ITO Electrode Surface for Studying Cytochrome b562 Electrochemistry

An interface of gold nanoshells (GNSs) was constructed on the surface of the 3-aminopropyl triethoxysilane (APTES) modified ITO glass substrates by a simple self-assemble method to form the GNSs-coated ITO electrode. UV-vis spectroscopy, scanning electron microscopy (SEM), and cyclic voltammetry were used to characterize the GNSs interface architectures. SEM and UV-vis spectroscopy showed that an interconnected and stable GNSs interface was formed on the APTES modified ITO glass substrate. The cytochrome b562 (Cyt b562) was selected to observe electron transfer reactions of redox protein at the GNSs-coated ITO electrodes. Quasi-reversible electrochemistry of Cyt b562 was obtained and its electrochemical behaviors were discussed.

Stability and Folding Kinetics of Structurally Characterized Cytochrome c-b562,

The four-helix-bundle protein fold can be constructed from a wide variety of primary amino acid sequences. Proteins with this structure are excellent candidates for investigations of the relationship between folding mechanism and topology. The folding of cytochrome b(562), a four-helix-bundle heme protein, is hampered by heme dissociation. To overcome this complication, we have engineered a variant of cytochrome b(562) (cyt c-b(562)) featuring a c-type linkage between the heme and the polypeptide chain. The replacement of the native cyt b(562) leader sequence in this protein with that of a c-type cytochrome (cyt c(556)) led to high yields of fully matured and correctly folded cyt c-b(562). We have determined the X-ray crystal structure of cyt c-b(562) at 2.25 A and characterized its physical, chemical, and folding properties. These measurements reveal that the c-type linkage does not perturb the protein fold or reduction potential of the heme group. The covalent attachment of the porphyrin to the polypeptide does, however, produce a substantial change in protein stability and folding kinetics.

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary.

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Two distinct quinone-modulated modes of antimycin-sensitive cytochrome b reduction in the cytochrome bc1 complex

Reduction of cytochrome b-560 (analogous to cyt b-562 of mitochondria) via an antimycin-sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center Q B site enters the Q pool and, via the Q c site, equilibrates with cytochrome b-560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc 1 complex perhaps involving a direct Q B-Q C site interaction. This latter reaction is suppressed by occupancy of the Q C site, not only by antimycin but by ubiquinol and ubiquinone.