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T Mogi

Publications and source records attributed to T Mogi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Probing a role of subunit IV of the Escherichia coli bo-type ubiquinol oxidase by deletion and cross-linking analyses.

Subunit IV of the Escherichia coli bo-type ubiquinol oxidase is a 12-kDa membrane protein encoded by the cyoD gene and is conserved in the bacterial heme-copper terminal oxidases. To probe the functional role of subunit IV, we carried out deletion analysis and chemical cross-linking experiments with a homobifunctional and cleavable reagent. Spectroscopic properties of the mutant oxidases suggest that the C-terminal two-third (Val45 to His109) containing helices II and III is essential for the functional expression of the oxidase complex and for the CuB binding to the heme-copper binuclear center in subunit I. Cross-linking studies indicate that subunit IV is in close vicinity to subunit III. Based on these observations, we propose that subunit IV is present in a cleft formed by subunits I and III and assists the CuB binding to subunit I during biosynthesis or assembly of the oxidase complex.

Amino Acid Sequence↗
PubMed1996

EPR study of NO complex of bd-type ubiquinol oxidase from Escherichia coli.

The heme axial ligands of bd-type ubiquinol oxidase of Escherichia coli were studied by EPR and optical spectroscopies using nitric oxide (NO) as a monitoring probe. We found that NO bound to ferrous heme d of the air-oxidized and fully reduced enzymes with very high affinity and to ferrous heme b595 of the fully reduced enzyme with low affinity. EPR spectrum of the 14NO complex of the reduced enzyme exhibited an axially symmetric signal with g-values at g = 2.041 and g = 1.993 and a clear triplet of triplet (or a triplet of doublet for the 15NO complex) superhyperfine structure originating from a nitrogenous proximal ligand trans to NO was observed. This EPR species was assigned to the ferrous heme d-NO complex. This suggests that the proximal axial ligand of heme d is a histidine residue in an anomalous condition or other nitrogenous amino acid residue. Furthermore, the EPR line shape of the ferrous heme d-NO was slightly influenced by the oxidation state of the heme b595. This indicates that heme d exists in close proximity to heme b595 forming a binuclear center. Another axially symmetric EPR signal with g-values at g(parallel) = 2.108 and g(perpendicular) = 2.020 appeared after prolonged incubation of the reduced enzyme with NO and was attributed to the ferrous heme b595-NO complex.

Binding Sites↗
PubMed1996

Infrared and EPR studies on cyanide binding to the heme-copper binuclear center of cytochrome bo-type ubiquinol oxidase from Escherichia coli. Release of a CuB-cyano complex in the partially reduced state.

Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CN-inhibited enzyme with excess sodium dithionite, a 12C-14N stretching vibration at 2146 cm-1 characteristic of the FeO3+ C=N CuB2+ bridging structure was quickly replaced with another stretching mode at 2034.5 cm-1 derived from the FeO2+ C=N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CN-inhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm-1. This 2169 cm-1 species could not be retained with a membrane filter (molecular weight cutoff = 10,000) and showed unusual cyanide isotope shifts and a D2O shift. These observations together with metal content analyses indicate that the 2169 cm-1 band is due to a CuB.CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CN-inhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the CuB center can be easily released upon cyanide-binding.

Anaerobiosis↗
PubMed1996

An endemic condition of biochemical hypoglycemia among male volunteers.

Biochemical hypoglycemia has attracted attention because it triggers accidents through errors in judgment by impairing cognitive function. Its prevalence and associated risk factors have remained unexplored in the area of occupational health. The present study was designed to investigate its epidemiological aspects in male volunteers. The study population of 76 male volunteers, 63 of whom were firefighters and 13 had other jobs, filled out questionnaires by interview covering 37 factors concerning age, physical parameters, past and current health problems, habits (smoking, drinking, and exercise), food preferences and life style. The participants were asked to measure their blood glucose levels 9 times over a representative workday and holiday and record any symptoms of hypoglycemia at the time of measurement. The body fat ratio of each participant was measured by the bioelectrical impedance analysis method. 22.4% of the participants (17/76) experienced biochemical hypoglycemia (less than 60 mg/dl) at the time of blood glucose monitoring. Those who experienced biochemical hypoglycemia had significantly lower peak, average and nadir blood glucose levels (p < 0.05; 115.1 +/- 18.7 mg/dl, 82.2 +/- 7.0 mg/dl, 52.2 +/- 5.6 mg/dl, respectively) than those who did not (129.9 +/- 26.7 mg/dl, 97.0 +/- 9.9 mg/dl, 75.9 +/- 9.7 mg/dl, respectively). No symptom other than sudden hunger (p < 0.05) was correlated with biochemical hypoglycemia. Lower lean body mass index (lean body mass/height2) was the only statistically significant contributory risk factor (p < 0.05, Odds ratio: 2.91, 95% confidence interval 1.29-6.65) by multiple logistic analysis. Mean blood glucose levels had a positive correlation with lean body mass index (P < 0.0001, r2 = 0.372). The present results suggest that biochemical hypoglycemia is a potential risk problem in occupational settings. Low L.B.M.I. males should be educated about the possibility of biochemical hypoglycemia whoever have experienced a feeling of sudden hunger. Such precautions should be highly recommended to workers under dangerous and/or tense conditions in order to prevent error accidents in occupational settings.

Adult↗
PubMed1996

Epidemiological study on hypoglycemia endemic to female nurses and other workers.

Hypoglycemia has been reported to occur frequently among individuals with health-related occupations, especially hospital nurses. In this study, we investigated the risk factors of hypoglycemia. Both female hospital nurses and other female workers participated in this study, which included questionnaires, and daily blood glucose monitoring. Responses from 3,415 workers were included for analysis. 2,489 workers reported that they had never had hypoglycemic episodes (symptom-free group), while 926 workers reported that they had experienced hypoglycemic symptoms more than once. Of these workers, 207 had had episodes within one year of the study and had experienced them more than once a month (symptomatic group). Thirty-one volunteers were recruited from the above two groups-21 from the latter group and 10 from the former group-and daily blood glucose levels were determined: 6 days for nurses to cover three shifts (dayshift, nightshift and midnightshift) and 2 days for other workers. Nine of 21 volunteers from the symptomatic group had biochemical hypoglycemic reading(s) (< 60 mg/dl) during the monitoring; however, none of 10 volunteers from the symptom-free group had such readings. These nine volunteers were found to have lower mean blood glucose levels (p < 0.05) and lower minimum blood glucose levels (p < 0.05) than other volunteers. Simple logistic analyses revealed multiple risk factors in the symptomatic group. By adjusting the confounding effects, these multiple risk factors were reduced into a small number of risk factors: occupation as a nurse, histories of hypotension and glucosuria, eating snacks, eating deserts, and insomnia. The present report clearly demonstrated that there are hypoglycemic-prone individuals among symptomatic female workers, who were shown to have multiple risk factors for hypoglycemic symptoms.

Adult↗
PubMed1996

[Multi-drug resistance of serovar E strains in clinically isolated Pseudomonas aeruginosa and analysis of genome pattern].

We analyzed the in vitro antibiotic susceptible pattern and serovar for 132 strains of pseudomonas aeruginosa (98 from inpatients and 34 from outpatients) isolated at Showa University hospital from September to December of 1993. The ratio of resistant strains was 38.6% for carbenicillin, 12.1% for piperacillin, 12.9% for ceftazidime, 12.1% for cefsulodin, 17.4% for cefoperazone, 9.1% for cefclidin, 24.2% for cefpirome, 7.6% for imipenem, 17.4% for amikacin, 16.7% for aztreonam, 89.4% for minocycline and 33.3% for ofloxacin. The incidence of resistance against minocycline, carbenicillin, ofloxacin was markedly high. Among the 132 tested strains of Pseudomonas aeruginosa, 16 were multi-drug resistant strains (i.e., resistant to more than 8 drugs out of 12 drugs), and 14 of the multi-drug resistant strains (87.5%) were isolated from inpatients. Multi-drug resistant strains were isolated most frequently from urine specimens. The antibiotics that remained effective against the multi-drug resistant strains included amikacin, imipenem, cefclidin and cefsulodin. The serovar of 116 strains (resistant to up to 7 drugs) was 27 for G, 24 for B, and 19 for E, and 15 of the 16 multi-drug resistant strains showed serovar E. Restriction enzyme SpeI digestion analysis (using pulse field electrophoresis) of P. aeruginosa having serovar E revealed common 650kb and 540kb bands in multi-drug resistant strains. The genome pattern was quoted for serovar E strain according to the restriction enzyme digestion pattern and was shown to be useful for tracing the route of infection.

Drug Resistance, Microbial↗
PubMed1995

Characterization of chimeric heme-copper respiratory oxidases using subunits I of Escherichia coli cytochrome b o and Halobacterium salinarium cytochrome aa3.

We constructed chimeric enzymes with the Escherichia coli cytochrome bo and the Halobacterium salinarium cytochrome aa3 through recombinant DNA techniques and investigated their spectroscopic and biochemical properties. Although most of the chimeras could not retain hemes in the molecule, the chimeric enzyme containing helix VII of subunit I of the H. salinarium cytochrome aa3 showed the spectral properties similar to those of the native E. coli oxidase, suggesting that both the low-spin heme b and the high-spin heme o are associated with the chimeric subunit I. However, CuB was absent in the chimera. Helix VII of subunit I of the H. salinarium cytochrome aa3 is 70% similar to the counterpart of the E. coli cytochrome bo and further contains two invariant histidines which serve as the CuB ligands. These results indicate that helix VII must be arranged properly relative to helix VI which provides the third CuB ligand.

Amino Acid Sequence↗
PubMed1995

Cyanide-binding site of bd-type ubiquinol oxidase from Escherichia coli.

We extended our investigation on the structure of the redox centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe. We found that addition of cyanide to the air-oxidized O2-bound enzyme caused appearance of an infrared C-N stretching band at 2161 cm-1 and concomitant disappearance of the 647 nm absorption band of the cytochrome d (Fe2+)-O2 species. Addition of cyanide to the air-oxidized CO-bound enzyme also resulted in disappearance of the 635 nm absorption band and the 1983.4 cm-1 C-O infrared band of the cytochrome d (Fe2+)-CO species. The resulting species had a derivative-shaped electron paramagnetic resonance signal at g = 3.15. Upon partial reduction with sodium dithionite, this species was converted partly to a transient heme d (Fe3+)-C = N species having an electron paramagnetic resonance signal at gz = 2.96 and a C-N infrared band at 2138 cm-1. These observations suggest that the active site of the enzyme has a heme-heme binuclear metal center distinct from that of the heme-copper terminal oxidase and that the treatment of the air-oxidized enzyme with cyanide resulted in a cyanide-bridging species with "heme d(Fe3+)-C = N-heme b595(Fe3+)" structure.

Binding Sites↗
PubMed1995

Stabilization of a semiquinone radical at the high-affinity quinone-binding site (QH) of the Escherichia coli bo-type ubiquinol oxidase.

Reaction of ubiquinone in the high-affinity quinone-binding site (QH) in bo-type ubiquinol oxidase from Escherichia coli was revealed by EPR and optical studies. In the QH site, ubiquinol was shown to be oxidized to ubisemiquinone and to ubiquinone, while no semiquinone signal was detected in the oxidase isolated from mutant cells that cannot synthesize ubiquinone. The QH site highly stabilized ubisemiquinone radical with a stability constant of 1-4 at pH 8.5 and the stability became lower at the lower pH. Midpoint potential of QH2/Q couple was -2 mV at pH 8.5 and showed -60 mV/pH dependence indicative of 2H+/2e- reaction. The Em was more negative than that of low-spin heme b above pH 7.0. We conclude that the QH mediates intramolecular electron transfer from ubiquinol in the low-affinity quinol oxidation site (QL) to low-spin heme b. Unique roles of the quinone-binding sites in the bacterial ubiquinol oxidase are discussed.

Benzoquinones↗
PubMed1995

CuB promotes both binding and reduction of dioxygen at the heme-copper binuclear center in the Escherichia coli bo-type ubiquinol oxidase.

A CuB-deficient mutant of the Escherichia coli bo-type ubiquinol oxidase exhibits a very low oxidase activity that is consistent with a decreased dioxygen binding rate. During the turnover, a photolabile reaction intermediate persists for a few hundred milliseconds, due to much slower heme o-to-ligand electron transfer. Thus, the lack of CuB seems to have endowed the mutant enzyme with myoglobin-like properties, thereby stabilizing the CO-bound form, too. Accordingly we conclude that CuB plays a pivotal role in preferential trapping and efficient reduction of dioxygen at the heme-copper binuclear center.

Carbon Monoxide↗
PubMed1995

Facilitated intramolecular electron transfer in the Escherichia coli bo-type ubiquinol oxidase requires chloride.

Previous flow-flash measurements using the bo-type ubiquinol oxidase of Escherichia coli have revealed that facilitated heme B-heme O intramolecular electron transfer initiated upon reaction of the fully-reduced enzyme with dioxygen proceeds with a rate constant higher than 5 x 10(4) s-1 at pH 7.4 and 20 degrees C. Depletion of chloride anions from the enzyme by HPLC performed in the present study considerably decreased the rate constant to approximately 700 s-1, but the reaction of either dioxygen or carbon monoxide at the binuclear center was not affected at all kinetically. These results strongly suggest that Cl- is essential in maintaining a subtle molecular structure around the heme B and heme O that enables facilitated intramolecular electron transfer. Furthermore, a series of absorption spectra of the enzyme collected on time scales from microseconds to milliseconds during its single turnover indicate that as heme-heme intramolecular electron transfer is retarded by depletion of Cl-, an alternative electron transfer pathway is invoked. We discuss a possible role of novel bound Cl- in electron transfer from bound quinol to the binuclear center to accomplish dioxygen reduction.

Carbon Monoxide↗
PubMed1995

A mouse model of a sudden death induced by noise exposure is useful to investigate human responses to physical stress.

Stress has become an increasingly important occupational health matter in Japan. We found that noise exposure, 6 kHz wave and its higher harmonics at the sound pressure level of 112 dB, induced typical audiogenic seizures in the MRL/l mouse strain. The seizures are often fatal. Wild running preludes to seizures. Inhibition of wild running by restricting animal's activities successfully prevented the seizures, suggesting that an excessive physical load is a necessary step. The present finding indicates that MRL/l mice may be a useful model for human sudden deaths after excessive work loads.

Acoustic Stimulation↗
PubMed1994

Molecular structure of redox metal centers of the cytochrome bo complex from Escherichia coli. Spectroscopic characterizations of the subunit I histidine mutant oxidases.

A site-directed mutagenesis study on the conserved subunit I histidines of the cytochrome bo complex in Escherichia coli identified ligands of the low spin heme B and CuB centers; however, the assignment of the proximal ligand of the high spin heme O was ambiguous (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). We have extended this work and characterized the metal centers in the purified histidine mutant oxidases by optical, EPR, and resonance Raman spectroscopies and by biochemical analysis. We found that the H284A and H333A oxidases contain two heme B molecules, which exhibit the gz = 2.99 low spin and cyanide-sensitive g perpendicular = 6 high-spin EPR signals, whereas the H419A oxidase contains only low spin heme B, which shows the gz component in a considerably higher magnetic field at g = 2.92. The CuB center was partially retained in the H284A oxidase but was almost completely lost in the H333A and H419A oxidases. Thus, we concluded that His419 is the proximal ligand of the high spin heme O and that His284 is located at the distal side of the high spin heme O. His284 plays an indispensable role in maintaining the structure of the Feo-CuB binuclear site suitable for the exogenous ligand bindings in the reduced state, since its substitution eliminated the CO binding activity. In addition, we found that His106 and His421 are in fact the axial ligands of the low spin heme B, although the H421A mutation perturbed the binuclear metal center seriously. Based upon experimental results with isotopic substitutions on iron in the oxidases, we assigned the Raman band at 208 cm-1 to the iron-histidine stretching mode (vFc(2+)-N(His)) of the wild-type ferrous cytochrome o, which was upshifted slightly by a loss of the CuB center in the H333A oxidase. A molecular structure of the metal centers and a possible mechanism of the electron transfer-coupled proton pumping in the cytochrome bo complex are proposed on the basis of our present findings.

Copper↗
PubMed1994

Structure-function studies on the ubiquinol oxidation site of the cytochrome bo complex from Escherichia coli using p-benzoquinones and substituted phenols.

To characterize the structural features of the quinol oxidation site (the QL site) of the cytochrome bo complex, a heme-copper respiratory oxidase in Escherichia coli, we carried out structure-inhibitory potency analyses using 7 p-benzoquinones and 33 substituted phenols. Their effects on its ubiquinol-1 oxidase activity were compared with those on the cytochrome bd complex in E. coli and on cytochromes o and alpha 1 in Acetobacter aceti. They showed similar structural properties of the QL site, although cytochrome o was more sensitive to 4-cyanophenols, suggesting a specific interaction of the hydrogen bond-accepting cyano group with the binding pocket. Replacing one of the methyl groups of 2,6-dimethyl-p-benzoquinone, which is the most potent competitive inhibitor, with an ethyl group markedly decreased the inhibitory activity, indicating that the QL site specifically recognizes one C = O group with two methyl groups as the ortho-substituents. In substituted phenols, ortho-chlorine substituents were the most effective in recognition, and the electron-withdrawing ability of the para-substituent determined an inhibitory potency, probably by stabilizing an anionic form. Based on these observations, we postulate that the QL site of the cytochrome bo complex asymmetrically recognizes exogenous ligands and that this property accounts for the sequential electron transfer from ubiquinols to the low-spin heme.

Benzoquinones↗
PubMed1994

Identification of a novel quinone-binding site in the cytochrome bo complex from Escherichia coli.

The cytochrome bo complex is a heme BO-type heme-copper quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as an electron transfer-linked proton pump. To study the protein-mediated electron transfer from substrates to metal centers, we carried out quantitative and qualitative analyses of a bound quinone in the purified oxidase and found that it has a novel high affinity ubiquinone-binding site distinct from the quinol oxidation site. Enzymatic and spectroscopic studies suggest that the quinone-binding site is located close to both the quinol oxidation site in subunit II and low-spin heme B in subunit I. The quinone-binding site of a bound ubiquinone-free oxidase was reconstituted with the potent quinol oxidation site inhibitor 2,6-dichloro-4-nitrophenol, which decreased the Vmax value of the ubiquinol-1 oxidase activity to one-fourth of the control activity. These results indicate that the quinone-binding site is essential for the catalytic functions of the cytochrome bo complex and mediates electron transfer from the quinol oxidation site to the low-spin heme.

Air↗
PubMed1994

Facilitated intramolecular electron transfer in cytochrome bo-type ubiquinol oxidase initiated upon reaction of the fully reduced enzyme with dioxygen.

Flow-flash and double-flash studies of the reaction of fully reduced bo-type quinol oxidase with oxygen have revealed that a single turnover of the enzyme proceeds much faster than mammalian cytochrome c oxidase. Facilitated intramolecular electron transfer in the bo-type oxidase with k > 5 x 10(4) s-1 at pH 7.4 and 20 degrees C is responsible for this fast turnover. The kinetics of this reaction indicates that the oxygen reduction does not require electron exchange between quinol oxidase molecules, each having three metal centers. Thus, a bound quinol in the fully reduced enzyme is suggested to be an electron source for complete reduction of dioxygen into water supplementing electrons provided by the metal centers. A single turnover of the quinol oxidase yields a novel spectral species with a Soret maximum at 415 nm corresponding to a 'pulsed' state of mammalian cytochrome c oxidase.

Animals↗
PubMed1994

Observation of the Fe-O2 and FeIV=O stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli.

Reaction intermediates in dioxygen reduction by the E. coli cytochrome bo-type ubiquinol oxidase were studied by time-resolved resonance Raman spectroscopy using the artificial cardiovascular system. At 0-20 microseconds following photolysis of the enzyme-CO adduct in the presence of O2, we observed the Fe-O2 stretching Raman band at 568 cm-1 which shifted to 535 cm-1 with the 18O2 derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa3-type cytochrome c oxidase. In the later time range (20-40 microseconds), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm-1. Since the 781 cm-1 band exhibited a downshift by 37 cm-1 upon 18O2 substitution, we assigned it to the FeIV=O stretching mode. This band is considered to arise from the ferryl intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (> 100 microseconds). The 361 cm-1 band showed the 16O/18O isotopic frequency shift of 14 cm-1 similar to the case of bovine cytochrome c oxidase reaction.

Cytochrome b Group↗