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.     

Fourier-transform infrared studies on conformation changes in bd-type ubiquinol oxidase from Escherichia coli upon photoreduction of the redox metal centers.

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli that does not belong to the heme-copper terminal oxidase superfamily. To explore unique protein structural changes associated with the reduction of the redox metal centers, we carried out Fourier-transform infrared and visible spectroscopic studies on cytochrome bd. For infrared measurements of a partially dehydrated thin sample solution, the air-oxidized enzyme was fully reduced by the intermolecular electron transfer of photo-excited riboflavin in the absence and presence of KCN, and redox difference spectra were calculated. Upon reduction, the bound cyanide was released from the heme b595-heme d binuclear center but remained in a protein pocket as a deprotonated form. Reduction of heme b558, heme b595, and heme d resulted in large changes in amide-I and protonated carboxylic CO-stretching vibrations and also a small change in the cysteine SH-stretching vibration. The location of the redox metal centers and the effects of cyanide suggest that these protein structural changes occur at the heme-binding pockets near the protein surface. Systematic site-directed mutagenesis and time-resolved FTIR studies on cytochrome bd will facilitate an understanding of the unique molecular mechanisms for dioxygen reduction and delivery of chemical protons to the active center at the atomic level.     

Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy

The mechanism of the dioxygen (O(2)) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O(2)-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (Delta UbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F(I) intermediate possessing ferryl-oxo heme o, cupric Cu(B), and ferric heme b is converted to the F(II) intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F(II) intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu(B) and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.     

Substrate-induced conformational changes of melibiose permease from Escherichia coli studied by infrared difference spectroscopy

Fourier transform infrared difference spectroscopy has been used to obtain information about substrate-induced structural changes of the melibiose permease (MelB) from Escherichia coli reconstituted into liposomes. Binding of the cosubstrate Na(+) gives rise to several peaks in the amide I and II regions of the difference spectrum Na(+).MelB minus H(+).MelB, that denote the presence of conformational changes in all types of secondary structures (alpha-helices, beta-sheets, loops). In addition, peaks around 1400 and at 1740-1720 cm(-1) are indicative of changes in protonation/deprotonation or in environment of carboxylic groups. Binding of the cosubstrate Li(+) produces a difference spectrum that is also indicative of conformational changes, but that is at variance as compared to that induced by Na(+) binding. To analyze the following transport steps, the melibiose permease with either H(+), Na(+), or Li(+) bound was incubated with melibiose. The difference spectra obtained by subtracting the spectrum cation.MelB from the respective complex cation.melibiose.MelB were roughly similar among them, but different from those induced by cation binding, and more intense. Therefore, major conformational changes that are induced during melibiose binding/substrate translocation, like those denoted by intense peaks at 1668 and 1645 cm(-)(1), are similar for the three cotransporting cations. Changes in the protonation state and/or in the environment of given carboxylic residues were also induced by melibiose-MelB interaction in the presence of cations.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

Defining the structural domain of subunit II of the heme-copper terminal oxidase using chimeric enzymes constructed from the Escherichia coli bo-type ubiquinol oxidase and the thermophilic Bacillus caa(3)-type cytochrome c oxidase.

To probe the location of the quinol oxidation site and physical interactions for inter-subunit electron transfer, we constructed and characterized two chimeric oxidases in which subunit II (CyoA) of cytochrome bo-type ubiquinol oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa(3)-type cytochrome c oxidase from thermophilic Bacillus PS3. In pHNchi5, the C-terminal hydrophilic domain except a connecting region as to transmembrane helix II of CyoA was replaced with the counterpart of CaaA, which carries the Cu(A) site and cytochrome c domain. The resultant chimeric oxidase was detected immunochemically and spectroscopically, and the turnover numbers for Q(1)H(2) (ubiquinol-1) and TMPD (N,N, N',N'-tetramethyl-p-phenylenediamine) oxidation were 28 and 8.5 s(-1), respectively. In pHNchi6, the chimeric oxidase was designed to carry a minimal region of the cupredoxin fold containing all the Cu(A) ligands, and showed enzymatic activities of 65 and 5.1 s(-1), and an expression level better than that of pHNchi5. Kinetic analyses proved that the apparent lower turnover of the chimeric enzyme by pHNchi6 was due to the higher K(m) of the enzyme for Q(1)H(2) (220 microM) than that of cytochrome bo (48 microM), while in the enzyme by pHNchi5, both substrate-binding and internal electron transfer were perturbed. These results suggest that the connecting region and the C-terminal alpha(1)-alpha(2)-beta(11)-alpha(3) domain of CyoA are involved in the quinol oxidation and/or physical interactions for inter-subunit electron transfer, supporting our previous proposal [Sato-Watanabe, M., Mogi, T., Miyoshi, H., and Anraku, Y. (1998) Biochemistry 37, 12744-12752]. The close relationship of E. coli quinol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacillus was also indicated.     

Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the DeltayhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in gamma-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.     

Use of an azido-ubiquinone derivative to identify subunit I as the ubiquinol binding site of the cytochrome d terminal oxidase complex of Escherichia coli

The radiolabeled, photoreactive azido-ubiquinone derivative (azido-Q), 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl-[3H]octyl)- 1,4-benzoquinone, was used to investigate the active site of ubiquinol oxidase activity of the cytochrome d complex, a two-subunit terminal oxidase of Escherichia coli. The azido-Q, when reduced by dithioerythritol, was shown to support enzymatic oxygen consumption by the cytochrome d complex that was 8% of the rate observed with ubiquinol-1. This observation provided the rationale behind further studies of the possible photoinactivation and labeling of the active site by this azido-Q. Ten min of photolysis of the purified cytochrome d complex in the presence of the azido-Q resulted in a 60% loss of the ubiquinol-1 oxidase activity. Uptake of the radiolabeled azido-Q by the cytochrome d complex was correlated to the photoinactivation of the ubiquinol-1 oxidase activity. Both increased linearly during the first 4 min of photolysis and reached 90% of the maximum within 10 min. Photolysis times longer than 10 min resulted in no increase in the maximum of 2 mol of azido-Q incorporated per mol of enzyme. The rate of azido-Q uptake by subunit I, but not subunit II, correlated well with the rate of loss of ubiquinol oxidase activity. Use of ubiquinol-0, which is not oxidized by the enzyme, to competitively inhibit radiolabeling of nonspecific binding sites, resulted in a significant decrease (42%) of azido-Q labeling of subunit II while it did not affect the labeling of subunit I. After photolysis for 4 min, the ratio of radiolabeled azido-Q in subunits I to II of the complex was 4.3 to 1.0. These observations support the conclusion that the ubiquinol substrate binding site is located on subunit I of the cytochrome d complex.     

Investigations into the polymorphism of lipid A from lipopolysaccharides of Escherichia coli and Salmonella minnesota by Fourier-transform infrared spectroscopy

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5 degrees and 58 degrees C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41 degrees, 46 degrees, 44 degrees and 47 degrees C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4'-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52 degrees C for E. coli lipid A). The analysis of greater than CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22 degrees, 30 degrees, 27 degrees and 25.5 degrees C (first transition) and at about 34 degrees, 42 degrees, 38.5 degrees and 40.5 degrees C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: Up to about 30 degrees C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of greater than CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product.

Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)     

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Cytochrome bo₃ is the major respiratory oxidase located in the cytoplasmic membrane of Escherichia coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (Q_H). The quinone bound at the Q_H site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the Q_H site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1–10, does not displace ubiquinone-8 from the Q_H site. The data clearly support the existence of a second binding site for ubiquinone, the Q_L site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1–10 may compete for binding with quinol at the Q_L site while, at the same time, preventing formation of the ubisemiquinone at the Q_H site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.     

Classification and identification of bacteria by Fourier-transform infrared spectroscopy.

This study describes a computer-based technique for classifying and identifying bacterial samples using Fourier-transform infrared spectroscopy (FT-IR) patterns. Classification schemes were tested for selected series of bacterial strains and species from a variety of different genera. Dissimilarities between bacterial IR spectra were calculated using modified correlation coefficients. Dissimilarity matrices were used for cluster analysis, which yielded dendrograms broadly equated with conventional taxonomic classification schemes. Analyses were performed with selected strains of the taxa Staphylococcus, Streptococcus, Clostridium, Legionella and Escherichia coli in particular, and with a database containing 139 bacterial reference spectra. The latter covered a wide range of Gram-negative and Gram-positive bacteria. Unknown specimens could be identified when included in an established cluster analysis. Thirty-six clinical isolates of Staphylococcus aureus and 24 of Streptococcus faecalis were tested and all were assigned to the correct species cluster. It is concluded that: (1) FT-IR patterns can be used to type bacteria; (2) FT-IR provides data which can be treated such that classifications are similar and/or complementary to conventional classification schemes; and (3) FT-IR can be used as an easy and safe method for the rapid identification of clinical isolates.     

Substrate-induced conformational changes in Escherichia coli arginyl-tRNA synthetase observed by 19F NMR spectroscopy

The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl-tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA(Arg) are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.     

Ligand interactions and protein conformational changes of phosphopyridoxyl-labeled Escherichia coli phosphoenolpyruvate carboxykinase determined by fluorescence spectroscopy.

Escherichia coli phosphoenolpyruvate (PEP) carboxykinase catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2. The interaction of the enzyme with the substrates originates important domain movements in the protein. In this work, the interaction of several substrates and ligands with E. coli PEP carboxykinase has been studied in the phosphopyridoxyl (P-pyridoxyl)-enzyme adduct. The derivatized enzyme retained the substrate-binding characteristics of the native protein, allowing the determination of several protein-ligand dissociation constants, as well as the role of Mg2+ and Mn2+ in substrate binding. The binding affinity of PEP to the enzyme-Mn2+ complex was -8.9 kcal.mol-1, which is 3.2 kcal.mol-1 more favorable than in the complex with Mg2+. For the substrate nucleotide-metal complexes, similar binding affinities (-6.0 to -6.2 kcal.mol-1) were found for either metal ion. The fluorescence decay of the P-pyridoxyl group fitted to two lifetimes of 5.15 ns (34%) and 1.2 ns. These lifetimes were markedly altered in the derivatized enzyme-PEP-Mn complexes, and smaller changes were obtained in the presence of other substrates. Molecular models of the P-pyridoxyl-E. coli PEP carboxykinase showed different degrees of solvent-exposed surfaces for the P-pyridoxyl group in the open (substrate-free) and closed (substrate-bound) forms, which are consistent with acrylamide quenching experiments, and suggest that the fluorescence changes reflect the domain movements of the protein in solution.     

Probing conformational changes of proteins by quantitative second-derivative infrared spectroscopy

Probing protein conformational changes plays a crucial role in protein structure and function studies. However, the lack of efficient biophysical techniques makes it difficult to obtain the distinct behaviors of different secondary structure elements in a protein upon perturbation. This paper presents a discussion of the two major problems, the effect of sidelobes and different half-width at half-height (HWHH) values, encountered in quantitative second-derivative infrared (QSD-IR) spectroscopy and introduces the development of two criteria for checking the validity of the results obtained using the QSD-IR method. It was found that neither the sidelobes nor the HWHH significantly affected the quantitative result of protein conformational changes by using poly-l-lysine and hemoglobin as model proteins. A case study of bovine serum albumin (BSA) thermal aggregation suggested that the thermal transition of BSA was a process involving sequential events, and the two helical components were found to have a distinct response to heat perturbation. These results were confirmed by two-dimensional infrared correlation spectroscopy and by results in literature, suggesting that the QSD-IR method might be a potentially powerful tool to probe the distinct response of different secondary structures to perturbation.     

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.     

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.     

Domain structure and ATP-induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis

Effector caspases-3, -6 and -7 are responsible for producing the morphological features associated with apoptosis, such as DNA fragmentation. The present study demonstrates that a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-6, induces apoptosis in MCF-7 cells, which lack caspase-3. Apoptosis was accompanied by DNA fragmentation and the activation of caspase-7, but not caspases-3 and -6. Inhibition of caspase-7 activity reduced the extent of apoptosis induced, indicating that activation of caspase-7 is involved in the mechanism by which PBOX-6 induces apoptosis in MCF-7 cells. This study suggests that caspase-3 is not necessarily essential for DNA fragmentation and the morphological changes associated with apoptosis.     

Redox-induced conformational changes within the Escherichia coli NADH ubiquinone oxidoreductase (complex I): An analysis by mutagenesis and FT-IR spectroscopy

The proton-pumping NADH:ubiquinone oxidoreductase couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. This process is suggested to be accompanied by conformational changes of the enzyme that may be monitored by redox-induced FT-IR difference spectroscopy. Signals observed in the amide I range are partially attributed to local rearrangements that occur as an electrostatic response to the redox reactions of the FeS clusters. In addition, conformational changes can be reported that depend on pH and at the same time can be perturbed by site-directed mutagenesis of residue E67 on subunit B (the bacterial homologue of the mitochondrial PSST subunit). This residue is located in the vicinity of the cluster N2. Re-evaluating these previous data we here discuss a mechanism, by which the redox reaction of N2 induces conformational changes possibly leading to proton translocation.