Fusako Takeuchi

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

BackgroundCytochrome 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.MethodsEffects 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 β-mercarptopropionic acid by inclusion of positively-charged poly-L-lysine. On the other hand, static midpoint potentials were measured under a similar condition.ResultsTitration 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.ConclusionsPresent 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.

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.

Cytochrome b561 is not fatty acylated but acetylated at amino terminus in chromaffin vesicle membranes: an approach for the identification of posttranslational modification of transmembrane proteins.

Summary. We examined the nature of the posttranslational modification of bovine cytochrome b561, a membrane-spanning protein and an essential component of neuroendocrine secretory vesicles. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed two populations in the partially digested fragments of cytochrome b561, which were obtained by controlled treatment of cytochrome b561-proteoliposomes with trypsin. One population, containing the posttranslationally modified amino-terminal region, showed molecular masses which were by about 40 Da larger than the theoretical molecular masses. The other population, without the modified amino-terminal region, showed a reasonable matching with the theoretical masses. This result suggested that the posttranslational modification occurred only in the amino-terminal region. The amino-terminal peptide was isolated by tryptic peptide mapping followed by treatment with acylamino-acid-releasing enzyme. Amino acid sequence and MALDI-TOF-MS analyses of the amino-terminal peptide showed that the initial Met residue was acetylated. There was no other posttranslational modification in the amino-terminal region, such as covalent fatty acylation through an ester linkage to Ser or Thr residues.

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).

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.

Interaction of Tail-anchored Proteins with Liposomes in Different Cholesterol Content: Initial Steps for the Fabrication of Artificial Neuroendocrine Vesicles

A group of membrane proteins endowed with a single C-terminal hydrophobic domain capable of insertion into lipid bilayer is known as tail-anchored (TA) proteins. We analyzed the integration of the TA domain of holo-form of human cytochrome b5 (HCYTb5) into protein-free liposomes different in a lipid composition. The integration of holo-b5 occurred efficiently into membranes with low cholesterol content and, once incorporated, the proteoliposomes were very stable. For membranes with high cholesterol content, incorporation efficiency was not so high. However, carbonate-extraction followed by discontinuous sucrose gradient showed that HCYTb5 was tightly incorporated into both types of vesicle membranes. Tryptic digestion of proteoliposomes containing HCYTb5 and free HCYTb5 showed a clear difference in the cleavage sites. The former cleavage site was identical to that occurred for native holo-b5 upon treatment of ER microsomes with trypsin

Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b5 reductase for the binding of β-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).

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.

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.

Properties of Human Tumor Suppressor 101F6 Protein as a Cytochrome b561 and Its Preliminary Crystallization Trials

Identification of the physiological roles and elucidation of the molecular mechanisms involving tumor suppressor genes and their gene products are important for a more comprehensive understanding of cancer pathogenesis. Since the Knudson’s statistical studies on retinoblastoma, neuroblastoma, and pheochromocytoma, which led to the conclusion that the occurrence of these tumors fits a two-mutation model (Knudson, 1971; Knudson & Strong, 1972), it became recognized that there were some genes that function to inhibit tumor development. The model stated that tumorigenesis results when there are genetic alterations such as deletions and mutations in both alleles of a gene in a cell (Knudson, 1971; Knudson & Strong, 1972). A tumor suppressor gene may have one or more functions related to cell division and differentiation, extracellular communication, tissue formation or senescence (Hollingsworth & Lee, 1991). Several regions of the human chromosome 3 have been identified as susceptible sites for homozygous deletions and mutations that may lead to inactivation of one or more tumor suppressor genes. A particular tumor suppressor gene candidate 101F6 is located within a narrow 630-kb region on chromosome 3p.21.3, called LUCA (lung cancer region) (Lerman & Minna, 2000; Zabarovsky et al., 2002). Interestingly, the 101F6 protein is expressed in normal lung bronchial epithelial cells and fibroblasts but is lost in most lung cancers (Ohtani et al., 2007). Previous studies have shown that forced expression of the 101F6 gene via adenoviral vector-mediated gene transfer (Ji et al., 2002) or via nanoparticle injection (Ohtani et al., 2007) caused the inhibition of tumor growth in non-small cell lung cancer cells in vitro and in vivo. The treated cancer cells were also found to accumulate ascorbate (AsA) when incubated in a medium containing AsA (Ohtani et al., 2007). Apoptosis and autophagy of the cancer cells were reportedly to be enhanced by the treatment and were postulated to be caused by the synergistic action of the 101F6 gene and AsA though the mechanism of the action is still not clear (Ohtani et al., 2007).