Functional Assembly of Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome in a model organism , Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state.

Multistep Changes in Amyloid Structure Induced by Cross-Seeding on a Rugged Energy Landscape.

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils.

Direct measurements of ferric reductase activity of human 101F6 and its enhancement upon reconstitution into phospholipid bilayer nanodisc.

We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration-dependent accelerations of its reaction with reasonable values of and .

9-Aryl-3-aminocarbazole as an Environment- and Stimuli-Sensitive Fluorogen and Applications in Lipid Droplet Imaging.

Environment-sensitive luminophoric molecules have played an important role in the fields of smart materials, sensing, and bioimaging. In this study, it was demonstrated that depending on the substituents, 9-aryl-3-aminocarbazoles can display aggregation-induced emission and solvatofluorochromism, and the operating mechanism was clarified. The application of these compounds to lipid droplet imaging and fluorescent probes for cysteamine was demonstrated.

Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5-Coordinate CO-Heme.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC-CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC-NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5-coordinate CO-heme, but the detailed mechanism remains unresolved.

Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe-O Complex.

Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate N-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from Deinococcus radiodurans was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 μs.

Photosensitization of Fluorofuroxans and Its Application to the Development of Visible Light-Triggered Nitric Oxide Donor.

Nitric oxide (NO) is an endogenous signaling molecule used in multiple biochemical processes. The development of switchable NO donors that deliver an NO payload under spatiotemporal control harbors many medicinal benefits. Previously, 4-fluorofuroxans were found to function as a UV light-induced NO donor under physiological conditions based on the photoinduced isomerization mechanism; however, the isomerization of fluorofuroxans with longer wavelength light is desired for further application into living systems.

The Radical S-Adenosyl-L-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds.

The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation.

A pulse radiolysis study of the dynamics of ascorbic acid free radicals within a liposomal environment.

The dynamics of free-radical species in a model cellular system are examined by measuring the formation and decay of ascorbate radicals within a liposome with pulse radiolysis techniques. Upon pulse radiolysis of an N2O-saturated aqueous solution containing ascorbate-loaded liposome vesicles, ascorbate radicals are formed by the reaction of OH(·) radicals with ascorbate in unilamellar vesicles exclusively, irrespective of the presence of vesicle lipids. The radicals are found to decay rapidly compared with the decay kinetics in an aqueous solution.

Electron transfer reactions of candidate tumor suppressor 101F6 protein, a cytochrome b561 homologue, with ascorbate and monodehydroascorbate radical.

The candidate tumor suppressor 101F6 protein is a homologue of adrenal chromaffin granule cytochrome b561, which is involved in the electron transfer from cytosolic ascorbate to intravesicular monodehydroascorbate radical. Since the tumor suppressor activity of 101F6 was enhanced in the presence of ascorbate, it was suggested that 101F6 might utilize a similar transmembrane electron transfer reaction. Detailed kinetic analyses were conducted on the detergent-solubilized recombinant human 101F6 for its electron transfer reactions with ascorbate and monodehydroascorbate radical by stopped-flow and pulse radiolysis techniques.

Roles of conserved Arg(72) and Tyr(71) in the ascorbate-specific transmembrane electron transfer catalyzed by Zea mays cytochrome b561.

Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys(83) on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg(72) and Tyr(71).

Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b(561) homologue.

Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b(561) family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b(561). Haem content analysis indicated presence of two haems B per molecule.

Interaction of modified tail-anchored proteins with liposomes: effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b₅.

A group of membrane proteins having a single COOH-terminal hydrophobic domain capable of post-translational insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b(5) (Hcytb5) into ER membranes, we produced and purified various membrane-bound forms of Hcytb5 with their heme b-bound, in which various truncated forms of NH(2)-terminal bovine opsin sequence were appended at the COOH-terminus of the native form. We analyzed the integration of the TA-domains of these forms onto protein-free liposomes.

Direct electrochemical analyses of human cytochromes b5 with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials.

Background: Cytochrome b5 performs central roles in various biological electron transfer reactions, where difference in the redox potential of two reactant proteins provides the driving force. Redox potentials of cytochromes b5 span a very wide range of ~400 mV, in which surface charge and hydrophobicity around the heme moiety are proposed to have crucial roles based on previous site-directed mutagenesis analyses.

Methods: Effects of mutations at conserved hydrophobic amino acid residues consisting of the heme pocket of cytochrome b5 were analyzed by EPR and electrochemical methods.

Importance of the conserved lysine 83 residue of Zea mays cytochrome b(561) for ascorbate-specific transmembrane electron transfer as revealed by site-directed mutagenesis studies.

Cytochromes b(561), a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic transmembrane alpha-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b(561) obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z.

Inhibition of electron acceptance from ascorbate by the specific N-carbethoxylations of maize cytochrome b561: a common mechanism for the transmembrane electron transfer in cytochrome b561 protein family.

Cytochromes b(561) constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane alpha-helices and two haem groups. Of particular interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b(561) heterologously expressed in Pichia pastoris cells.

Functional expression and characterization of human 101F6 protein, a homologue of cytochrome b561 and a candidate tumor suppressor gene product.

A highly hydrophobic protein with six transmembrane structure that is coded by the candidate tumor suppressor gene 101F6 located in the human chromosome 3p.21.3 and a possible member of the cytochrome b 561 protein family was expressed, purified, and characterized in its functional form for the first time.

Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b(5) reductase for the binding of beta-NADH.

Well-conserved three consecutive Pro residues (Pro247-249) in the NADH-binding subdomain of NADH-cytochrome b(5) reductase were proposed to form a basal part of the NADH-binding site. To investigate the structural and mechanistic roles of these residues, we expressed site-directed mutants for a soluble domain of the porcine enzyme where each of the residues was replaced with either Ala or Leu residue, respectively, using a heterologous expression system in Escherichia coli. Six mutants (P247A, P247L, P248A, P248L, P249A, and P249L) were produced as a fusion protein containing a 6xHis-tag sequence at the NH(2)-terminus and were purified to homogeneity with a stoichiometric amount of bound FAD.

Characterization of heme-coordinating histidyl residues of an engineered six-coordinated myoglobin mutant based on the reactivity with diethylpyrocarbonate, mass spectrometry, and electron paramagnetic resonance spectroscopy.

A genetically engineered porcine myoglobin triple mutant (H64V/V68H/H93A) (VHA-Mb) contains 6 non-axial His residues (His24, His36, His48, His81, His82, and His119) besides two candidate axial His residues (His68 and His97). Although previous resonance Raman study on the ferric VHA-Mb were not conclusive for its coordination structure, present EPR parameters of the ferric VHA-Mb were consistent with bis-imidazole coordination of His68/His97. We further investigated the reactivity of these possible His ligands with diethylpyrocarbonate (DEPC) to clarify the coordination structure and their protonation states in ferric form.

Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer.

Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four different transmembrane alpha-helices. Although cytochromes b(561) residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer.

Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings.

We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e.

Selective perturbation of the intravesicular heme center of cytochrome b561 by cysteinyl modification with 4,4'-dithiodipyridine.

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with EPR signals at g(z) = 3.69 and 3.14 and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase.

Cytochrome b561 protein family: expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses.

Cytochrome b561 family was characterized by the presence of "b561 core domain" that forms a transmembrane four helix bundle containing four totally conserved His residues, which might coordinate two heme b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL W on b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D (fungi), E (animals/TSF), F (plants+DoH), and G (SDR2).

Planarian peptidylglycine-hydroxylating monooxygenase, a neuropeptide processing enzyme, colocalizes with cytochrome b561 along the central nervous system.

Planarians are one of the simplest animal groups with a central nervous system. Their primitive central nervous system produces large quantities of a variety of neuropeptides, of which many are amidated at their C terminus. In vertebrates, peptide amidation is catalyzed by two enzymes [peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxylglycine alpha-amidating lyase] acting sequentially.

Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay.

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with different midpoint potentials (+150 and +60 mV) and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of oxidized cytochrome b(561) with diethylpyrocarbonate caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of ascorbate during the treatment. Present EPR analyses showed that, upon the treatment, the g(z) = 3.