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

Among seven homologs of cytochrome b561 in a model organism C. elegans, 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 Pichia pastoris 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. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

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. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

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 K M and V max. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe2+-specific colorimetric chelator. 101F6 in nanodisc state showed higher efficiency for FAC than in detergent micelle state.

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. In this study, we examined the mechanism under the condition including BAY 41-2272, 2'-deoxy-3'-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO-heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO-heme, as assessed by IR spectral measurements. The two types of CO-hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO-heme, and subsequently followed by the slower second step leading a partial conversion of the 6-coordinate CO-heme to the 5-coordinate CO-heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5-coordinate CO-heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO-heme. This mechanism provides a new aspect on the activation of sGC by CO.

Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to FeII-O2 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. Subsequently, ferrous heme reacted with O2 to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 108 M-1 s-1. In the tetrahydrofolate (H4F)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 103 s-1. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H4F, this step did not proceed, suggesting that H4F was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s-1. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s-1). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates.

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. Herein, we report the use of photosensitizers in the photochemical isomerization of fluorofuroxan, enabling the use of visible light to induce isomerization. Among the tried photosensitizers, anthraquinone derivatives showed a good sensitizing ability to isomerize 4-fluorofuroxan to 3-fluorofuroxan using visible light. This new phenomenon was applied to the synthesis of a water-soluble anthraquinone-fluorofuroxan all-in-one molecule, which demonstrated promising NO-releasing ability using 400-500 nm irradiation. A high level of control is displayed with "on" and "off" NO-release functionality suggesting that photosensitizer-furoxan hybrids would make valuable donors. Furthermore, unlike most furoxans, NO is released in the absence of thiol cofactor.

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. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5'-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate.

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. The distinct radical reaction kinetics in the vesicles and in bulk solution are characterized, and the kinetic data are analyzed.

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. The reduction of oxidized 101F6 with ascorbate was found to be independent of pH in contrast to those observed for chromaffin granule and Zea mays cytochromes b561 in which both cytochromes exhibited very slow rates at pH 5.0 but faster at pH 6.0 and 7.0. The absence of the inhibition for the electron acceptance from ascorbate upon the treatment with diethyl pyrocarbonate suggested that 101F6 might not utilize a "concerted proton/electron transfer mechanism". The second-order rate constant for the electron donation from the ascorbate-reduced 101F6 to the pulse-generated monodehydroascorbate radical was found to be 5.0 × 10(7) M(-1 )s(-1), about 2-fold faster than that of bovine chromaffin granule cytochrome b561 and about five times faster than that of Zea mays cytochrome b561, suggesting that human 101F6 is very effective for regenerating ascorbate from monodehydroascorbate radical in cells. Present observations suggest that 101F6 employs distinct electron transfer mechanisms on both sides of the membranes from those of other members of cytochrome b561 protein family.

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). Removal of a positive charge at Arg(72) did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr(71) with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr(71) and Arg(72) with ascorbate. However, their mechanistic roles were distinctly different from that of Lys(83), as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons.

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. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b(561). Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g(z) = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at g(z) = 3.65 being overlapped with that of g(z) = 3.75. The rhombic EPR signal at g(z) = 3.16 previously seen in other cytochromes b(561) was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b(561) where the 'concerted H(+)/e(-) transfer mechanism' at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b(561).

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. The integration occurred efficiently even in the presence of a small amount of sodium cholate and, once incorporated, such proteoliposomes were very stable. The mode of the integration was further analyzed by treatment of the proteoliposomes with trypsin either on the extravesicular side or on the luminal side. LC-MS analyses of the trypsin digests obtained from the proteoliposomes indicated that most of the C-terminal hydrophilic segment of the native Hcytb5 were exposed towards the lumen of the vesicles and, further, a significant part of the population of the extended C-terminal hydrophilic segments of the modified Hcytb5 were exposed in the lumen as well, suggesting efficient translocation ability of the TA-domain without any assistance from other protein factors. Present results opened a route for the use of the C-terminal TA-domain as a convenient tool for the transport of proteins as well as short peptides into artificial 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. Cyclic voltammetry of the heme-binding domain of human cytochrome b5 (HLMWb5) and its site-directed mutants was conducted using a gold electrode pre-treated with ß-mercarptopropionic acid by inclusion of positively-charged poly-L-lysine. On the other hand, static midpoint potentials were measured under a similar condition. RESULTS: Titration of HLMWb5 with poly-L-lysine indicated that half-wave potential up-shifted to -19.5 mV when the concentration reached to form a complex. On the other hand, midpoint potentials of -3.2 and +16.5 mV were obtained for HLMWb5 in the absence and presence of poly-L-lysine, respectively, by a spectroscopic electrochemical titration, suggesting that positive charges introduced by binding of poly-L-lysine around an exposed heme propionate resulted in a positive shift of the potential. Analyses on the five site-specific mutants showed a good correlation between the half-wave and the midpoint potentials, in which the former were 16~32 mV more negative than the latter, suggesting that both binding of poly-L-lysine and hydrophobicity around the heme moiety regulate the overall redox potentials. CONCLUSIONS: Present study showed that simultaneous measurements of the midpoint and the half-wave potentials could be a good evaluating methodology for the analyses of static and dynamic redox properties of various hemoproteins including cytochrome b5. The potentials might be modulated by a gross conformational change in the tertiary structure, by a slight change in the local structure, or by a change in the hydrophobicity around the heme moiety as found for the interaction with poly-L-lysine. Therefore, the system consisting of cytochrome b5 and its partner proteins or peptides might be a good paradigm for studying the biological electron transfer reactions.

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. mays cytochrome b(561) as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys(83) residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys(83) might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser(118) and Trp(122) do not have major roles for the redox events on the intravesicular side.

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. Pre-treatment of cytochrome b(561) with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His(86), His(159) and Lys(83), leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two haem centres did show only slight decreases upon the treatment, suggesting that changes in the midpoint potentials were not the major cause of the inhibition. Present results indicated that Zea mays cytochrome b(561) conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H(+)/e(-) transfer mechanism and that the specific N-carbethoxylation of haem axial His(86) that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys(83) might be important for an initial step(s) of the fast electron acceptance from ascorbate.

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. Mutations were each confirmed for the purified proteins by MALDI-TOF mass spectrometry. Steady-state kinetic analyses for NADH:ferricyanide reductase and NADH:cytochrome b(5) reductase acitivities were conducted for all the mutants. Substitution of Pro247 with Leu residue was found to significantly decrease k(cat) with slight increase in K(m) for the physiological electron donor NADH. However, K(m) values for the electron acceptors (both cytochrome b(5) and ferricyanide) of P247L were found to be decreased significantly. Such changes were not observed for P247A or other four mutants. These results suggested that Pro247 among the three consecutive Pro residues has the most important role for the formation of a binding site cavity and that only a slight change in the side-chain volume at this residue from Ala to Leu residue affected the electron transfer reaction from NADH and, further, on the recognition of ferricytochrome b(5).

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. We found that the non-axial His residues were easily modified with a low concentration of DEPC based on UV spectral changes and MALDI-TOF-MS analyses. On the other hand, the two candidate axial His ligands were protected from the modification due to a limited steric exposure of their imidazoles to solvent, the Fe(3+)-N(epsilon2) coordination bond, and the protonation of N(delta1) by forming a hydrogen bond with their immediate surroundings. However, once N-carbethoxylation occurred at N(epsilon2) of His97, resulting in a disruption of the heme Fe(3+)-N(epsilon2) coordination bond, it facilitated the second N-carbethoxylation to take place at N(delta1) of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. These phenomena were consistent with the bis-His68/His97 coordination. Further, these were not observed at all for cytochrome b(561), a transmembrane di-heme containing protein responsible for the ascorbate-specific transmembrane electron transfer, where only a specific N(delta1)-carbethoxylation of axial His occurred at a low concentration of DEPC, leading to an inhibition of the electron acceptance from ascorbate without a release of the heme. These distinct results might be related to a specific physiological mechanism being operative at the cytosolic heme center of cytochrome b(561).

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. The protein was heterologously expressed in methylotrophic yeast Pichia pastoris as a fusion protein containing a C-terminal thrombin-specific sequence and an 8-His residue tag. Purification was achieved by ion exchange chromatography on DEAE-Sepharose and affinity chromatography on Ni-NTA-Sepharose. SDS-PAGE analysis revealed a single protein band with an estimated molecular weight of 26 kDa, while Western blot and MALDI-TOF-MS analysis confirmed the presence of the cytochrome b561 specific sequence in the protein. The 101F6 protein was found to be reducible by ascorbate efficiently and to have two midpoint potentials at +89.5 and +13.1 mV, slightly lower than the corresponding values of +155 and +62 mV, respectively, of bovine adrenal cytochrome b 561, despite a lower conservation of the putative ascorbate binding site sequence in the 101F6 protein. The "modified motif 1" sequence unique in 101F6 protein may be responsible for other molecular functions, such as protein-protein interactions, in the endoplasmic membranes.

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. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants.