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.
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.
Packed with vitamins: The formation and decay of ascorbate free radicals within liposomes are examined by using pulse radiolysis techniques. These radicals are found to decay rapidly compared with those in aqueous solution.
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 b₅ (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 NH₂-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.
Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b561 family. Purified 101F6 protein expressed in Pichia ...pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b561. 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 b561. Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at gz = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at gz = 3.65 being overlapped with that of gz = 3.75. The rhombic EPR signal at gz = 3.16 previously seen in other cytochromes b561 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 b561 where the 'concerted H+/e- transfer mechanism' at the cytosolic haem centre was blocked by specific N epsilon -carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b561.
Cytochromes b₅₆₁ 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 α-helices. Although cytochromes b₅₆₁ 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 b₅₆₁ has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the Nδ₁ 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.
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 b5 (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 NH2-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.
Cytochrome b561 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 β-hydroxylase. Treatment of oxidized cytochrome b561 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 gz = 3.69 heme species was converted to a non-ascorbate-reducible form, although its gz-value showed no appreciable change. The treatment had no effect on the other heme (the gz = 3.13 species). Raman data indicated that the two heme b centers adopt a six-coordinated low-spin state, in both the reduced and oxidized forms. There was no significant effect of diethylpyrocarbonate-treatment on the Raman spectra of either form, but the reducibility by ascorbate differed significantly between the two hemes upon the treatment. The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the diethylpyrocarbonate-treated cytochrome b561 when ascorbate was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of ascorbate and, thereby, increases both the reduction rate and the final reduction level of the heme center on the intravesicular side of the diethylpyrocarbonate-treated cytochrome. These results further clarify the physiological role of this heme center as the electron donor to the monodehydroascorbate radical.
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 O
to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 10
M
s
. In the tetrahydrofolate (H
F)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 10
s
. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H
F, this step did not proceed, suggesting that H
F 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
. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s
). 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.
Cytochrome b₅₆₁ from bovine adrenal chromaffin vesicles contains two hemes b with EPR signals at gsubscript z = 3.69 and 3.14 and participates in transmembrane electron transport from extravesicular ...ascorbate to an intravesicular monooxygenase, dopamine {szligbeta}-hydroxylase. Treatment of purified cytochrome b₅₆₁ in an oxidized state with a sulfhydryl reagent, 4,4'-dithiodipyridine, caused the introduction of only one 4-thiopyridine group per b₅₆₁ molecule at either Cys57 or Cys125. About half of the heme centers of the modified cytochrome were reduced rapidly with ascorbate as found for the untreated sample, but the final reduction level decreased to approximately65%. EPR spectra of the modified cytochrome showed that a part of the gsubscript z = 3.14 low-spin EPR species was converted to a new low-spin species with gsubscript z = 2.94, although a considerable part of the heme center was concomitantly converted to a high-spin g = 6 species. Addition of ascorbate to the modified cytochrome caused the disappearance or significant reduction of the EPR signals at gsubscript z = 3.69 and 3.14 of low-spin species and at g = 6.0 of the high-spin species, but not for the gsubscript z approximately 2.94 species. These results suggested that the bound 4-thiopyridone at either Cys57 or Cys125 affected the intravesicular heme center and converted it partially to a non-ascorbate-reducible form. The present observations suggested the importance of the two well-conserved Cys residues near the intravesicular heme center and implied their physiological roles during the electron donation to the monodehydroascorbate radical.
Cytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The ...purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine β-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine β-hydroxylase.
