Biomedical subjects

T Mogi

Publications and source records attributed to T Mogi.

At least 73 records · Page 4Linked to original sources

PubMed1994

An Escherichia coli cyoE gene homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase.

The cyoE gene of the Escherichia coli bo-type quinol oxidase operon (cyoABCDE) has been previously shown to encode heme O synthase. To demonstrate a catalytic role of a cyoE homologue (the caaE gene) in the gene cluster for caa3-type cytochrome c oxidase of thermophilic Bacillus PS3, we have carried out genetic complementation analysis using the chimeric operon cyoABCD-caaE and heme O synthase assay using the CaaE-overproduced E. coli membranes. We found that the caaE gene encodes a thermotolerant heme O synthase which provides an intermediate for heme A biosynthesis.

Alkyl and Aryl Transferases↗

PubMed1994

Resonance Raman and Fourier transform infrared studies on the subunit I histidine mutants of the cytochrome bo complex in Escherichia coli. Molecular structure of redox metal centers.

The cytochrome bo complex is a heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. Site-directed mutagenesis studies were greatly successful in identifying ligands of the redox centers in the oxidase (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). In the present study, resonance Raman and Fourier transform infrared spectroscopies were applied to the subunit I histidine mutant oxidases, and the active-site structure was studied in detail. In the dithionite-reduced state, the wild-type oxidase and the His-284-->Ala and His-333-->Ala mutant oxidases showed two Raman lines (v3) at approximately 1492 and 1472 cm-1, which are attributable to low- and high-spin heme components, respectively. The Ala substitutions at His-106 and His-421 reduced the low-spin line, whereas the His-419-->Ala mutation eliminated the high-spin line. This indicates that His-106 and His-421 are the axial ligands of the low-spin heme B, while His-419 acts as a proximal ligand of the high-spin heme O. The v(Fe-CO) stretching frequency in the wild-type oxidase down-shifted from 523 to 498 cm-1 in the CuB-deficient mutant oxidase (His-333-->Ala), while the v(C-O) stretching frequency up-shifted from 1960 to 1970 cm-1. These frequency differences were ascribed to the contribution from the reduced CuB at the binuclear center. It may indicate that delocalization of electrons at the CuB center toward the heme O iron is facilitated by a bridge ligand structure at the binuclear center and is essential for driving dioxygen reduction chemistry, and therefore, redox-coupled proton pumping.

Alanine↗

PubMed1994

Molecular structure of a heme-copper redox center of the Escherichia coli ubiquinol oxidase: evidence and model.

Biochemical, spectroscopic, and molecular biological studies on bacterial respiratory oxidases in the last decade have greatly increased our understanding of a molecular structure of the metal centers which catalyze the dioxygen reduction chemistry. Based upon the latest physicochemical and molecular biological evidence and theoretical consideration of a folding mechanics of membrane proteins, we propose here the tertiary structure of the heme-copper metal center of the Escherichia coli bo-type ubiquinol oxidase, the cytochrome bo complex. The molecular mechanism of electron transfer-coupled proton pumping in the heme-copper respiratory oxidases is reviewed on the basis of this predicted model.

Amino Acid Sequence↗

PubMed1994

Biosynthesis and functional role of haem O and haem A.

Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases. A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.

Alkyl and Aryl Transferases↗

PubMed1993

Identification of the functional domains in heme O synthase. Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli.

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is encoded by the cyoABCDE operon. Recently, we have demonstrated that heme O at the high-spin heme-binding site is essential for redox-coupled proton pumping by the oxidase and suggested that the cyoE gene encodes a novel enzyme for heme O biosynthesis, protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). This study was focused to define the catalytic domain(s) of the CyoE protein via a site-directed mutagenesis approach. We have individually substituted 40 amino acid residues including 22 invariant residues with alanines and found that 23 mutant oxidases were nonfunctional and exhibited a specific loss of the CO binding activity at the site of the high-spin heme. Characterizations of the purified D65A, Y120A, and W172A mutant oxidases, which represent the mutations of different topological domains, revealed that their defects are attributable to substitution of protoheme IX for heme O present in the high-spin heme-binding site. Based on the above observations, we suggest that the conserved amino acid residues present in the cytoplasmic loops II/III and IV/V are part of the catalytic center of heme O synthase.

Alkyl and Aryl Transferases↗

PubMed1993

In vitro heme O synthesis by the cyoE gene product from Escherichia coli.

The cytochrome bo complex is a heme-copper terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and contains low spin heme B, high spin heme O and CuB as the redox metal centers in subunit I. Based on site-directed mutagenesis studies on the cyoE gene in the cytochrome bo operon, we have postulated that the cyoE gene encodes a protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). The present study demonstrates that the CyoE protein is localized in the cytoplasmic membrane and that the CyoE-overproduced membranes efficiently catalyze a conversion of exogenous ferrous protoheme IX and farnesyl diphosphate to heme O in the presence of divalent cations such as Mg2+ or Ca2+. Thus, the cyoABCDE operon in E. coli encodes not only subunits of the cytochrome bo complex but also heme O synthase that is specifically required for functional expression of the bo-type quinol oxidase. Heme O seems to be an intermediate in heme A biosynthesis.

Alkyl and Aryl Transferases↗

PubMed1993

Cytochrome d axial ligand of the bd-type terminal quinol oxidase from Escherichia coli.

Using various spectroscopic techniques, we studied the structure of the dioxygen reduction site of the bd-type terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli. Resonance Raman and FT-IR spectroscopies identified the v(Fe(2+)-CO) and v(C-O) stretching frequencies at 471 and 1980.7 cm-1, respectively, at the cytochrome d center of the dithionite-reduced CO-bound enzyme. The CO ligation in the cytochrome bd complex is considerably different from those of the heme-copper terminal oxidases. Anaerobic addition of NO to the air-oxidized enzyme caused an exchange of cytochrome d-bound dioxygen with NO leading to an appearance of cytochrome d-NO EPR signal. But there is no superhyperfine structure originating from the cytochrome d proximal 14N ligand in the central resonance of the NO EPR signal. These results suggest that cytochrome d axial ligand of the cytochrome bd complex is likely a histidine residue in an anomalous condition or other than a histidine residue and, therefore, the molecular structure around the dioxygen-binding site is different from that of the heme-copper terminal oxidases.

Binding Sites↗

PubMed1993

Structure of the heme-copper binuclear center of the cytochrome bo complex of Escherichia coli: EPR and Fourier transform infrared spectroscopic studies.

The cytochrome bo complex is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. To clarify the structural differences of the binuclear reaction center between the cytochrome bo complex and the mitochondrial cytochrome c oxidase, a combined study using EPR and Fourier transform infrared spectroscopies was carried out. The EPR spectrum of the highly purified cytochrome bo complex in the air-oxidized state showed a broad EPR signal (peak g* = 3.7) from an integer spin system. This confirms the existence of the spin-spin exchange-coupled binuclear site, in which the Feo3+ and CuB2+ centers were bridged by an unknown ligand (X). Binding of azide at the binuclear site as an ionic modulator weakened the strength of the spin-spin exchange coupling and thus caused a narrowing of the broad EPR signal. Binding of another modulator, formate, at the binuclear site caused the formation of EPR signals at g' = 12 and 2.7, which are very similar to those observed for cytochrome c oxidase. Cyanide replaced the bridging ligand (X) to form an Feo(3+)-C-N-CuB2+ structure in which strong spin-spin exchange coupling is expected, leading to a complete EPR-invisible state. Infrared evidence (a 2146 cm-1 C-N stretching band for the cyanide complex and a 2041 cm-1 azide antisymmetric stretching band for the azide complex) supported the theory that these ligands form bridging structures at the binuclear center, as previously observed for cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Azides↗

PubMed1993

Energy restriction suppresses microsomal Ca(2+)-ATPase activities in various organs in C57BL/6 female mice in both euthermic and torpor states.

C57BL/6 female mice were fed a daily control diet (n = 5, 3.9 g/day, 95 kcal/week) or ER diet (n = 5, 2.3 g/day, 48 kcal/week) at 1800 h from 6 weeks of age. Telemetry, conducted at 6 months of age, confirmed that all ER mice entered daily torpor (core body temperature less than 31 degrees C) for 6.63 +/- 2.34 h/day while control mice were euthermic (> 35 degrees C). In vitro activities of microsomal Ca(2+)-ATPase were determined in the brain, liver, salivary gland and kidney from these mice at 6 months of age. Assays were performed at three incubation temperatures of 37 degrees C, 31 degrees C and 25 degrees C. In assays at 37 degrees C, the activities of Ca(2+)-ATPase in the brain and salivary gland from ER mice were lower than those in corresponding organs from control mice. The suppression became profound as the incubation temperature decreased. On the other hand, at 37 degrees C Ca(2+)-ATPase activities in the liver and kidney from ER mice were not lower than those in corresponding organs from control mice, but decreased significantly at low temperatures. Microsomal Ca(2+)-ATPase activities thus appeared to be reduced in ER mice, although it remains unknown whether the present results represent reduced in vivo capacities to regulate cytosolic Ca2+ concentrations.

Animals↗

PubMed1993

Effects of mutagenetic substitution of prolines on the rate of deprotonation and reprotonation of the Schiff base during the photocycle of bacteriorhodopsin.

Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions of Pro-50, Pro-91 and Pro-186 on the deprotonation and reprotonation kinetics of the Schiff base (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate that Pro-50 and Pro-91, as well as Pro-186, are important for the proton-pumping function of bR.

Bacteriorhodopsins↗

PubMed1992

Heme O biosynthesis in Escherichia coli: the cyoE gene in the cytochrome bo operon encodes a protoheme IX farnesyltransferase.

The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme O present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme O. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel enzyme, protoheme IX farnesyltransferase, essential for heme O biosynthesis.

Alkyl and Aryl Transferases↗

PubMed1992

Identification of heme and copper ligands in subunit I of the cytochrome bo complex in Escherichia coli.

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli (Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3368-3374) and functions as a proton pump. It belongs to the heme-copper oxidase superfamily with the aa3-type cytochrome c oxidases in mitochondria and aerobic bacteria. In order to identify ligands of hemes and copper, we have substituted eight conserved histidines in subunit I by alanine and, in addition, His-106, -284, and -421 by glutamine and methionine. Western immunoblotting analysis showed that all the mutations do not affect the expression level of subunit I in the cytoplasmic membrane, indicating that these histidines are not crucial for its stability. A single copy expression vector carrying a single mutation at the invariant histidines, His-106, His-284, His-333, His-334, His-419, and His-421, of subunit I was unable to support the aerobic growth of a strain in which the chromosomal terminal oxidase genes (the cyo and cyd operons) have been deleted. The same mutations caused a complete loss of ubiquinol oxidase activity of the partially purified enzymes. Spectroscopic analysis of mutant oxidases in the cytoplasmic membrane revealed that substitutions of His-106 and -421 specifically eliminated a 563.5 nm peak of the low spin heme and that replacements of His-106, -284, and -419 reduced the extent of the CO-binding high spin heme. These spectroscopic properties of mutant oxidases were further confirmed with partially purified preparations. Atomic absorption analysis showed that substitutions of His-106, -333, -334, and -419 eliminated CuB almost completely. Based on these findings, we conclude that His-106 and -421 function as the axial ligands of the low spin heme and His-284 is a possible ligand of the high spin heme. His-333, -334, and -419 residues are attributed to the ligands of CuB. We present a helical wheel model of the redox center in subunit I, which consists of the membrane-spanning regions II, VI, VII, and X, and discuss the implications of the model.

Aerobiosis↗

PubMed1992

Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin.

The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.

Bacteriorhodopsins↗

PubMed1992

[Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96].

It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.

Aspartic Acid↗

PubMed1991

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin.

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

Bacteriorhodopsins↗

PubMed1991

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

Amino Acid Sequence↗

PubMed1991

Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle.

The rates are determined for the deprotonation and reprotonation of the protonated Schiff base (PSB) as well as of formation and decay of the UV transient in the photocycle of seven bacteriorhodopsin (bR) mutants in which Arg-7, 82, 164, 175, 225, or 227 are replaced by glutamine and Arg-134 by cysteine. The results show that all these mutations increase the rate of deprotonation of the PSB compared to ebR, (wild-type bacteriorhodopsin expressed in Escherichia coli) greatly increase the rate of the reprotonation of the SB (Schiff base) in the case of the Arg-164 and Arg-175 mutations and dramatically decrease this rate in the case of the Arg-227 mutation. Temperature studies on the latter mutant suggest that the observed change in its rate of reprotonation is due to large decrease in the energy and entropy of activation, similar to those observed for Asp-96 mutations (Miller, A. and D. Orsterhelt. 1990. Biochim. Biophys. Acta. 1020:57-64). These results suggest that the reprotonation process is changed to a proton diffusion-controlled mechanism in the Arg-227 mutant due to a change in the structure of the proton channel. The absorption intensity ratio (AUV/AMslow) of each arginine mutant relative to that of ebR is found to be similar to that for native purple membrane (PM) except for the Arg-227 mutant where it is greatly reduced, and for the Arg-82 mutant where it is not observed, suggesting that both Arg-227 and Arg-82 residues somehow play roles in inducing the UV transient absorption. All the above results are discussed in terms of the model for the structure of bR proposed by Henderson, R., J.M. Baldwin, T.A. Ceska, F. Zemlin, E. Beckmann, and K.H. Downing. (1990. J. Mol. Biol. 213:899-929).

Amino Acid Sequence↗

PubMed1990

Transcriptional regulation of the cytochrome b562-o complex in Escherichia coli. Gene expression and molecular characterization of the promoter.

Transcriptional regulation of the cyo operon coding for a terminal oxidase, the cytochrome b562-o complex, of the aerobic respiratory chain of Escherichia coli was studied using a chromosomal operon fusion technique with the lacZ gene. Expression of the cyo gene was found to be subject to catabolite repression and also to control by the oxygen concentration. Information on the mechanisms of these regulations was obtained by nucleotide sequencing of the regulatory region, which corresponds to the 5'-flanking region of the cyoA gene. A typical promoter sequence and two noteworthy composite structural features, that is, two potential catabolite gene activator protein-binding sites and a region of hyphenated dyad symmetry were found. The transcription start point was identified by primer extension analysis, confirming the promoter site in the nucleotide sequence. The homology search of the nucleotide sequence in the region of hyphenated dyad symmetry showed a conserved nucleotide sequence in six oxygen-regulated genes examined, which suggests that this is the consensus sequence for the regulation by oxygen.

Amino Acid Sequence↗