Combination treatment with statins and bezafibrate induces myotoxicity via inhibition of geranylgeranyl pyrophosphate biosynthesis and Rho activation in L6 myoblasts and myotube cells.

Statins and fibrates are frequently used to treat hyperlipidemia; however, these drugs may have adverse effects such as rhabdomyolysis. The incidence of rhabdomyolysis due to fibrates and statins is low (0.0028-0.0096%) when administered as monotherapy, however it increases to 0.015-0.021% when the drugs are used in combination. The mechanism underlying myotoxicity induced by the combination of statins and fibrates is yet unclear. Here, we investigated the mechanisms underlying induced myotoxicity in rat myoblasts L6 and differentiated L6 cells (myotubes) using a combination of statins and fibrates. We found that cell death induced by a combination of fluvastatin or simvastatin with bezafibrate or fenofibrate in L6 myoblasts and myotubes was mediated by inhibition of geranylgeranyl pyrophosphate (GGPP) production. Additionally, the drug combination inhibited Rho activation in L6 myoblasts and myotube cells. In L6 myoblasts, the combination of statins and bezafibrate enhanced p27 expression and induced G1 arrest and apoptosis. Furthermore, combined treatment suppressed Akt activation and enhanced Bim expression in L6 myotubes but did not affect extracellular regulated protein kinase 1/2 activation. These results suggested that combined administration of statins and fibrates induced death of L6 myoblasts and myotube cells by inhibiting GGPP biosynthesis and Rho pathway activation. Supplementation with GGPP may be therapeutically beneficial for preventing myotoxicity associated with combined statin and fibrates treatment.

Effect of proton pump inhibitors on the development of hypomagnesemia induced by panitumumab.

Panitumumab, a therapeutic agent for unresectable advanced/recurrent colorectal cancer, is a human IgG2 monoclonal antibody that binds to and inhibits the activity of the epidermal growth factor receptor (EGFR). The onset of hypomagnesemia is a known side effect of anti-EGFR inhibitors, including panitumumab, and it is thought that inhibition of reabsorption of Mg in renal tubules is one of the causes. In addition, recent reports have shown that long-term administration of proton pump inhibitors (PPIs) reduces serum magnesium levels. Therefore, in this study, 102 patients who received oral PPIs treated with panitumumab were classified into a PPI combination group and a PPI non-combination group, and the effect of PPIs on the development of grade 2 or higher hypomagnesemia was investigated. The incidence of hypomagnesemia in the PPI combination group (46.9%, 15/32) was higher than that in the PPI non-combination group (25.7%, 18/70). A comparison of the backgrounds of the two groups of patients showed a significant difference in serum albumin levels. PPI administration was significantly associated with panitumumab-induced hypomagnesemia development when adjusted for known risk factors, serum albumin level, renal function, and oral magnesium oxide tablets in Cox proportional hazards regression analysis (hazard ratio 2.09; 95% confidence interval 1.03-4.22; P =0.040). These results indicate that detailed monitoring of serum magnesium levels is recommended for patients treated with panitumumab and co-administration of PPIs.

Impact of intraperitoneal chemotherapy after gastrectomy with positive cytological findings in peritoneal washings.

BACKGROUND: There is no standard treatment available for gastric cancer patients whose sole 'non-curative factor' is positivecytological findings in peritoneal washings (CFPW). The aim of this study was to examine the safety, pharmacokinetics and efficacy for free intraperitoneal cancer cells of intraperitoneal chemotherapy with paclitaxel after gastrectomy with en bloc D2 lymph node dissection in cases of gastric cancer with positive CFPW. METHODS: Ten patients with gastric cancer who underwent gastrectomy and systemic lymphadenectomy with D2 dissection, without any other non-curative factors besides positive CFPW, were treated with early postoperative intraperitoneal paclitaxel. Intra-chemotherapeutic toxicity and operative complications were measured using NCI-CTC version 3.0. Intraperitoneal and plasma paclitaxel concentrations were measured using a high-performance liquid chromatographic assay. RESULTS: Grade 3/4 toxic effects included anemia (20%) and neutropenia (10%) that required no treatment. Operative complications were, for example, superficial surgical site infections (10%) that were treated with antibiotics. No viable cancer cells were observed in the intra-abdominal fluid 24 h after intraperitoneal administration of paclitaxel. The intraperitoneal/plasma area under the drug concentration-time curve ratio was 2,003.3:1. CONCLUSION: Intraperitoneal chemotherapy with paclitaxel is a safe and effective treatment modality for free intraperitoneal cancer cells.

Evaluation of NT-pro BNP and CT-ANP as markers of concentric hypertrophy in dogs with a model of compensated aortic stenosis.

BACKGROUND: Serum C-terminal atrial natriuretic peptide (CT-ANP) and N-terminal pro B-type natriuretic peptide (NT-pro BNP) concentrations have not been measured serially in dogs with chronic pressure overload of the heart. HYPOTHESIS: We investigated whether serial evaluation of CT-ANP and NT-pro BNP concentrations is a useful guide to the risk of cardiac remodeling in dogs with a model of aortic stenosis. ANIMALS: Six male Beagles. METHODS: After anesthesia, the aorta was constricted with a polyester band and mean left ventricular systolic pressure (LVPs) was 50 mmHg above baseline. Echocardiographic and intracardiac catheter examinations and blood sampling were performed before surgery and 3 and 6 months after surgery. RESULTS: LVP and left ventricular end-diastolic pressure (LVEDP) were significantly higher at 6 months. Compared with baseline, end-diastolic intraventricular septum thickness (IVSd), left ventricular posterior wall thickness (LVPWd), and relative wall thickness (RWT) were significantly increased 3 and 6 months after aortic constriction. Serum CT-ANP concentrations were increased significantly at 3 months and serum NT-pro BNP concentrations were significantly higher 3 and 6 months after aortic constriction. Serum NT-pro BNP concentration was significantly correlated with LVEDP and IVSd whereas serum CT-ANP concentration was not correlated with any measurement. Stepwise regression analysis showed that LVEDP, IVSd, and RWT could predict serum NT-pro BNP. CONCLUSIONS AND CLINICAL IMPORTANCE: This study indicated the differential regulation of NT-pro BNP and CT-ANP concentrations during pressure overload. NT-pro BNP assay may be used as an additional screening method to stratify early-stage ventricular remodeling because of aortic constriction.

Induction of apoptosis in HL-60 cells treated with medicinal herbs.

In order to develop a new apoptosis inducer, we screened 22 crude drugs for their apoptosis-inducing activity. It was found that Glycyrrhiza uralensis, Cynomorium songaricum, Eucommia ulmoides, Phellodendron amurense, Cinnamomum cassia and Paeonia lactiflora induced the death of HL-60 cells. To investigate the mechanism of apoptosis induced by these six crude drugs, the mitochondrial transmembrane potential and the activity of caspase-3 were measured. Reduced mitochondrial transmembrane potentials within 12 hours after the administration of Glycyrrhiza uralensis, Cynomorium songaricum, Phellodendron amurense and Paeonia lactiflora, and within 24 hours after the administration of Eucommia ulmoides and Cinnamomum cassia were observed. All of the six apoptosis-inducing crude drugs increased caspase-3 activity within 12-36 hours after administration. After further examining the apoptosis-inducing activity of berberine, palmatine, panelofuroline and glycyrrhizin, which were the ingredients obtained from Phellodendron amurense, Glycyrrhiza uralensis and Paeonia lactiflora, it was found that only berberine could induce apoptosis. From these results, it was concluded that the apoptosis induced by the six crude drugs (Glycyrrhiza uralensis, Cynomorium songaricum, Eucommia ulmoides, Phellodendron amurense, Cinnamomum cassia and Paeonia lactiflora) occurred via the mitochondrial route and that the apoptosis-conducting mechanism acted through a cascade involving caspase-3.

Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.

Ascorbate inhibits the carbethoxylation of two histidyl and one tyrosyl residues indispensable for the transmembrane electron transfer reaction of cytochrome b561.

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. We found previously that treatment of oxidized cytochrome b(561) with diethyl pyrocarbonate caused specific N-carbethoxylation of three fully conserved residues (His88, His161, and Lys85) located at the extravesicular side. The modification lead to a selective loss of the electron-accepting ability from ascorbate without affecting the electron donation to monodehydroascorbate radical [Tsubaki, M., Kobayashi, K., Ichise, T., Takeuchi, F., and Tagawa, S. (2000) Biochemistry 39, 3276-3284]. In the present study, we found that these modifications lead to a drastic decrease of the midpoint potential of heme b at the extravesicular side from +60 to -30 mV. We found further that the O-carbethoxylation of one tyrosyl residue (Tyr218) located at the extravesicular side was significantly enhanced under alkaline conditions, leading to a very slow reduction process of the oxidized heme b with ascorbate. On the other hand, the presence of ascorbate during the treatment with diethyl pyrocarbonate was found to suppress the carbethoxylation of His88, His161, and Tyr218, whereas the modification level of Lys85 was not affected. Concomitantly, the final reduction level of heme b with ascorbate was protected, although the fast reduction phase was not fully restored. These results suggest that the two heme-coordinating histidyl residues (His88 and His161) are also a part of the ascorbate binding site. Tyr218 and Lys85 may have a role in the recognition/binding process for ascorbate and are indispensable for the fast electron transfer reaction.

Unusual spin state equilibrium of azide metmyoglobin induced by ferric corrphycene.

Myoglobin was reconstituted with the ferric complex of corrphycene, a novel porphyrin isomer with a rearranged tetrapyrrole array, to investigate the influence of porphyrin deformation on the equilibrium between high-spin (S = 5/2) and low-spin (S = 1/2) states in the azide derivative. The azide affinity, 2.5 x 10(4) M(-1), was 1 order of magnitude lower than the corresponding values of a reference myoglobin containing an electron-deficient diformylheme similar to the corrphycene. Analysis of the visible absorption spectrum over a range of 0-40 degrees C reveals that the population of high-spin iron is 76-82% at room temperature for azide metmyoglobin complexed with ferric corrphycene. The unusual predominance of the high-spin state was verified from the infrared spectrum of coordinating azide, where the high-spin peak at 2046 cm(-1) is 4-fold larger in intensity than the 2023 cm(-1) low-spin band. Electron paramagnetic resonance at 15 K further indicated that the iron-histidine bond is cleaved to form a five-coordinate derivative in some fraction of the myoglobin. The remarkable high-spin bias of the spin equilibrium at room temperature and cleavage of the iron-histidine bond at 15 K could be explained in terms of the contracted and trapezoidal metallo core that weakens the iron-histidine bond of azide metmyoglobin bearing corrphycene.

Diethyl pyrocarbonate modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence electron donation to monodehydroascorbate radical: identification of the modification sites by mass spectrometric analysis.

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate.

Probing molecular structure of dioxygen reduction site of bacterial quinol oxidases through ligand binding to the redox metal centers.

Cytochromes bo and bd are structurally unrelated terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli. The high-spin heme o-CuB binuclear center serves as the dioxygen reduction site for cytochrome bo, and the heme b595-heme d binuclear center for cytochrome bd. CuB coordinates three histidine ligands and serves as a transient ligand binding site en route to high-spin heme o one-electron donor to the oxy intermediate, and a binding site for bridging ligands like cyanide. In addition, it can protect the dioxygen reduction site through binding of a peroxide ion in the resting state, and connects directly or indirectly Tyr288 and Glu286 to carry out redox-driven proton pumping in the catalytic cycle. Contrary, heme b595 of cytochrome bd participate a similar role to CuB in ligand binding and dioxygen reduction but cannot perform such versatile roles because of its rigid structure.

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus.

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.

Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies.

Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers. To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).

Fluoride-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption and EPR spectroscopies.

Cytochrome bd-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli contains two hemes b (b558 and b595) and one heme d as redox metal centers. To clarify the structure of the reaction center, we analyzed the fully oxidized enzyme by visible and EPR spectroscopies using fluoride ion as a monitoring probe. The visible spectral changes upon fluoride-binding were typical of ferric iron-chlorine species, indicating heme d as a primary binding site. The negative peak at 645 nm in the difference spectrum indicates that heme b595 also provides the low-affinity fluoride-binding site. Fluoride-binding caused a complete disappearance from the EPR spectra of the low-spin signals ascribable to heme d and spectral changes in both rhombic and axial high-spin signals. After fluoride-binding, each component of the rhombic high-spin signal showed superhyperfine splitting arising from the interaction of the unpaired spin of the heme d iron with the nuclear magnetic moment of 19F. The axial high-spin species was converted to a new rhombic high-spin species assignable to heme b595-fluoride. The g = 2 component of this new species also gave 19F-superhyperfine splitting. These results indicate that both heme d and heme b595 can coordinate with a fluoride ion with different affinities in the fully oxidized state.

Fourier-transform infrared studies on azide-binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase.

Azide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier-transform infrared spectroscopy. Deconvolution analyses of infrared spectra of the azide (14N3)-inhibited air-oxidized form showed a major infrared azide antisymmetric stretching band at 2041 cm(-1). An additional band developed at 2062.5 cm(-1) during a longer incubation. Isotope substitutions with terminally 15N-labelled azides did not show a splitting of the major band, indicating that the geometry of the bound azide is mainly in a bridging configuration between high-spin heme o and CuB. The band at 2062.5 cm(-1) showed clear splittings upon substitution with the terminally 15N-labelled azides, indicating the Cu(2+)B-N=N=N structure. Partial reduction of the oxidase with beta-NADH in the presence of azide caused an appearance of new infrared bands at 2038.5 (major) and 2009 (minor) cm(-1). The former band also showed clear splittings in the presence of the terminally 15N-labelled azides, indicating that reduction of low-spin heme b alters the structure of the binuclear center leading to the Fe(3+)o-N=N=N configuration.

A clinicopathological investigation on superficial early invasive carcinomas of the colon and rectum.

Due to recent advances in endoscopic surgical techniques, it has now become possible to perform endoscopic resection of most early invasive carcinomas of the colon and rectum (EIC) even if the lesions have invaded the submucosa. In the present study, we investigated the microscopic characteristics of superficial EIC compared with protruding-type EIC, focusing particular attention on histological type, the presence or absence of vascular invasion, the extent of submucosal invasion, and other adverse prognostic factors, to establish appropriate treatment strategies. Our findings led us to conclude that: (1) most cases of EIC can be cured by endoscopic resection if their gross aspects are classified as type IIc, superficial depressed, or type IIa, superficial elevated; (2) colorectal resection with lymph node dissection should be performed first for type IIa + IIc EIC because these lesions are apt to be associated with a large number of adverse prognostic factors; (3) subsequent colorectal resection should be performed after initial endoscopic treatment of EIC if there are adverse prognostic indicators of metastasis in the endoscopically resected specimen, such as moderately or poorly differentiated adenocarcinoma, lymphatic invasion, venous invasion, or extensive submucosal invasion.

[p53 expression and response to hepatic intraarterial infusion chemotherapy in patients with colorectal cancer metastasis to liver].

We have reported that hepatic artery infusion (HAI) chemotherapy with 5-fluorouracil for unresectable colorectal liver metastasis may lead to improved overall survival for some patients compared with controls. The aim of this work is to determine whether p53 nuclear protein expression examined by immunohistochemical technique could predict the outcome of patients treated with HAI chemotherapy. No significant difference was found in the response rate (p > .99, Fisher's exact method.) and median survival time (12 months in patients with p53-positive tumors and 11 months with negative tumors). Our results indicate that HAI chemotherapy is an effective treatment for liver metastasis regardless of p53 nuclear protein expression.

Distinct roles of two heme centers for transmembrane electron transfer in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by pulse radiolysis.

The reaction of monodehydroascorbate (MDA) radical with purified cytochrome b561 from bovine adrenal chromaffin vesicles was investigated by the technique of pulse radiolysis. Radiolytically generated MDA radical oxidized rapidly the reduced form of cytochrome b561 to yield the oxidized form. Subsequently the oxidized form of cytochrome b561 was re-reduced by ascorbate in the medium. The second-order rate constants of the reaction of MDA radical were increased with decreasing pH, whereas a maximum of the second-order rate constant for the reaction with ascorbate was obtained around pH 6.8. At excess MDA radical to cytochrome b561 concentration, only half of the heme in cytochrome b561 was oxidized, indicating that only one of the two heme centers can react with MDA radical. On the other hand, when the reactions were examined using cytochrome b561 pretreated in a mild alkaline condition in the oxidized state, the cytochrome b561 could not be oxidized with MDA radical, suggesting that the heme center specific for the electron donation to MDA radical is selectively modified upon the alkaline treatment. These results suggest that the two heme b centers have distinct roles for the electron donation to MDA radical and the electron acceptance from ascorbate, respectively.

20beta-hydroxy-C21-steroid 20beta-oxidase activity of cytochrome P450c21 purified from bovine adrenocortical microsomes.

We showed previously that cytochrome P450c21 (CYP21A1) from bovine adrenocortical microsomes has a putative 20beta-oxidase activity for 20beta-hydroxyprogesterone leading to a conversion to progesterone [M. Tsubaki, K. Morimoto, S. Tomita, S. Miura, Y. Ichikawa, A. Miyatake, F. Masuya, H. Hori, Biochim. Biophys. Acta 1259 (1995) 89-98]. We have extended the investigation on the 20beta-oxidase activity of cytochrome P450c21. Combination of 17alpha, 20beta-dihydroxyprogesterone with purified cytochrome P450c21 in oxidized form caused an induction of a typical type I difference spectrum (a peak at 390 nm and a trough at 420 nm) with a spectral dissociation constant of 2.3 microM. EPR spectrum at low temperature (15 K) exhibited an appearance of a new low-spin signal at gz=2.42, gy=2.21, and gx=1.966 and an increase in intensity of the high-spin signal (g=8.06, 3.54) upon formation of the enzyme-steroid complex, as previously found for 17alpha-hydroxyprogesterone and 20beta-hydroxyprogesterone. The enzymatic activity for 17alpha, 20beta-dihydroxyprogesterone was confirmed in a reconstituted system consisting of cytochrome P450c21 and NADPH-cytochrome P450 reductase. 17alpha,20beta-Dihydroxyprogesterone was converted to 17alpha-hydroxyprogesterone via the 20beta-oxidase reaction. The high turnover numbers of the 20beta-oxidase activity for 20beta-hydroxy-c21-steroids suggests that this activity is likely to have some physiological roles. Cytochrome P450c21 and 20beta-hydroxysteroid dehydrogenase may regulate the androgen biosynthesis catalyzed by cytochrome P450c17alpha with controlling the concentration of 20beta-hydroxy-C21-steroids.

Structural basis for the electron transfer across the chromaffin vesicle membranes catalyzed by cytochrome b561: analyses of cDNA nucleotide sequences and visible absorption spectra.

We isolated cDNA clones for cytochromes b561 from sheep and porcine adrenal medullae using the RT-PCR technique. Comparison of the deduced amino acid sequences of various species showed that there are two fully-conserved regions in this cytochrome. In addition, one methionyl and six histidyl residues (potential heme ligands) are fully-conserved. Based on a plausible structural model in which a polypeptide spans the vesicle membranes six times and holds two heme B molecules, the first conserved sequence (69ALLVYRVFR77) is located on the extravesicular side of an alpha-helical segment and the second one (120SLHSW124) is located in an intravesicular loop connecting two alpha-helical segments, respectively. Consideration of the relative locations of the fully-conserved sequences, and the methionyl and histidyl residues in the model led to a proposal that the first and second conserved sequences are likely to form the binding sites for extravesicular ascorbic acid and intravesicular semidehydroascorbic acid, respectively. A mild alkaline-treatment of purified bovine cytochrome b561 in oxidized state led to a specific loss of an electron-accepting ability from ascorbic acid for a half of the heme center, suggesting a distinct role for each of the two hemes.

Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase.

Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines. This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I. Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp. Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity. Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center. On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases.