Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells.

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.

A third subunit in ancestral cytochrome c-dependent nitric oxide reductases.

Reduction of NO to N2O by denitrifiying bacteria is catalyzed either by a monomeric quinol-nitric oxide reductase (qNor) or by a heterodimeric cytochrome c-dependent nitric oxide reductase (cNor). In ancient thermophilic bacteria belonging to the Thermales and Aquificales phylogenetic groups, the cluster encoding the cNor includes a small third gene (norH), in addition to those encoding homologues to the subunits of a typical cNor (norC and norB). We show in Thermus thermophilus that the three genes are cotranscribed in a single mRNA from an inducible promoter. The isolation of individual nor mutants and the production in vivo of His-tagged NorH protein followed by immobilized-metal affinity chromatography (IMAC) allowed us to conclude that NorH constitutes a third subunit of the cNor from T. thermophilus, which is involved in denitrification in vivo, likely allowing more efficient electron transport to cNor.

The cytochrome oxidase superfamily of redox-driven proton pumps.

Most respiratory oxidases of eukaryotic and prokaryotic organisms are members of a superfamily of enzymes that couple the redox energy available from the reduction of molecular oxygen to the mechanism of pumping protons across the membrane. The recent applications of site-directed mutagenesis and of a variety of spectroscopic techniques have allowed major advances in our understanding of the structure and function of these proteins.

Active site rearrangement and structural divergence in prokaryotic respiratory oxidases.

Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.

Protein structure: proton-pumping oxidases.

The crystal structures of two cytochrome c oxidases, one bacterial and one mammalian, offer insights into their roles in oxygen chemistry and as proton pumps.

Some recent advances relating to prokaryotic cytochrome c reductases and cytochrome c oxidases.

Prokaryotic systems provide excellent experimental opportunities for exploring structure/function relationships for the complex, membrane-bound, multisubunit enzymes responsible for the reduction and subsequent oxidation of c-type cytochromes in respiratory or photosynthetic electron transport chains. Two points are made in this mini-review: (1) The eukaryotic and prokaryotic aa3-type cytochrome c oxidases are members of an apparently large superfamily of structurally related respiratory oxidases. This superfamily displays considerable variation in terms of the heme prosthetic groups (a or b) as well as the substrate oxidized (quinol or cytochrome c). The relationships among these enzymes help to facilitate explorations of how they work. (2) Molecular biology techniques can be used to generate intact, redox-active, water-soluble domains of membrane-bound subunits. These soluble domains can be used for detailed examination, including obtaining high resolution structure by NMR techniques or by X-ray crystallography. This approach is being used to study the soluble heme-binding domain of cytochrome c1 from the bc1 complex of Rhodobacter sphaeroides.

Specific ligand enhancement of the affinity of E. coli pyruvate oxidase for dipalmitoyl phosphatidylcholine.

Pyruvate oxidase (pyruvate: ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme isolated from Escherichia coli. The enzyme catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2, and is coupled to the E. coli electron traansport chain. In vitro, pyruvate oxidase activity is measured spectrophotometrically using ferricyanide as an electron acceptor. In the presence of dipalmitoyl phosphatidylcholine or a number of other phospholipids, or detergents, the enzymatic specific activity is enhanced about 25-fold. In this paper the interaction between pyruvate oxidase and dipalmitoyl phosphatidylcholine is examined. It is demonstrated that the presence of the ligands involved in catalysis has a substantial influence on the affinity between pyruvate oxidase and dipalmitoyl phosphatidylcholine. In the absence of the substrate (pyruvate) and cofactor (thiamin pyrophosphate) there is no detectable complex formation. However, when both ligands are present, a condition which results in the reduction of the flavoprotein, the interaction between the protein and phospholipid is greatly enhanced. It is clearly shown that the protein-lipid interaction is dramatically modulated by the ligands bound at the catalytic active site on the enzyme and/or by the oxidation-reduction state of the flavin.

The binding of a fluorescent activator 2-(N-decyl)aminonaphthalene-6-sulfonic acid to pyruvate oxidase.

E. coli pyruvate oxidase (pyruvate:ferricytochrome b1 oxidoreductase, EC 1.2.2.2) is a peripheral membrane flavoenzyme which has been purified to homogeneity. In vivo the oxidase resides on the inner surface of the cytoplasmic membrane and is coupled to the bacterial electron transport chain. In vitro, the purified oxidase requires lipids for full enzymatic activity. Previous studies have characterized the conformational and energetic coupling between the lipid-binding site(s) and the catalytic active site. The affinity of the enzyme for phospholipids and detergents is significantly enhanced when the flavoprotein is in the reduced form, i.e., in the presence of pyruvate and the required cofactor, thiamin pyrophosphate. The lipid-binding studies were hindered due to the complicating factor of the self-association of the substrate-reduced flavoprotein. In this paper, fluorescence techniques are employed to measure the binding of a detergent-like activator to the oxidase. The experiments are performed at much lower protein concentrations than previously employed, so that protein aggregation is not a problem. The chromophore on the activator, 2-(N-decyl)aminonaphthalene-6-sulfonic acid is effective at quenching the pyruvate oxidase intrinsic tryptophan fluorescence. Quenching titrations are used to obtain the binding isotherm. AT DNS concentrations less than 10(-5) M, the results show a larger amount of DNS binding to the reduced flavoprotein than to the oxidized form of the enzyme. This is the concentration range where DNS is an effective activator of the enzyme. This represents a class of binding sites specifically found on pyruvate oxidase and not apparent in other proteins such as lysozyme or aldolase. At the DNS concentration which is optimum for activation approx. 20 molecules of DNS are bound per enzyme tetramer in the absence of the substrate. The pyruvate-reduced form of the enzyme binds about 40--50 molecules of DNS per tetramer. Qualitatively, the results are similar to what was previously found for both sodium dodecyl sulfate and cetyl trimethylammonium bromide. However, in both these cases, the amount of bound detergent was nearly an order of magnitude less than the values obtained using DNS.

In vivo assembly of the cytochrome d terminal oxidase complex of Escherichia coli from genes encoding the two subunits expressed on separate plasmids.

The cytochrome d terminal oxidase complex is a heterodimer located in the cytoplasmic membrane of Escherichia coli. Subunit II of the cytochrome d terminal oxidase complex was expressed independently of subunit I of the complex. It was found that the polypeptide is produced and is associated with the cytoplasmic membrane in the absence of subunit I, and is not associated with any of the three cytochrome components of the complex. Oxidase activity and heme binding are restored when the subunit I is expressed in the same cells using a second compatible plasmid. It has been previously demonstrated that subunit I, expressed in the absence of subunit II, contains cytochrome b-558, one of the three heme prosthetic groups found in the oxidase. Association of the two other heme moieties, cytochromes b-595 and d, apparently requires the association of the two subunits, and must be a late step in the assembly of the membrane-bound protein. It was also shown that under heme-deficient conditions, the two polypeptide subunits are expressed and are associated with the cytoplasmic membrane.

Proton pumping by cytochrome oxidase: progress, problems and postulates.

The current status of our knowledge about the mechanism of proton pumping by cytochrome oxidase is discussed. Significant progress has resulted from the study of site-directed mutants within the proton-conducting pathways of the bacterial oxidases. There appear to be two channels to facilitate proton translocation within the enzyme and they are important at different parts of the catalytic cycle. The use of hydrogen peroxide as an alternative substrate provides a very useful experimental tool to explore the enzymology of this system, and insights gained from this approach are described. Proton transfer is coupled to and appears to regulate the rate of electron transfer steps during turnover. It is proposed that the initial step in the reaction involves a proton transfer to the active site that is important to convert metal-ligated hydroxide to water, which can more rapidly dissociate from the metals and allow the reaction with dioxygen which, we propose, can bind the one-electron reduced heme-copper center. Coordinated movement of protons and electrons over both short and long distances within the enzyme appear to be important at different parts of the catalytic cycle. During the initial reduction of dioxygen, direct hydrogen transfer to form a tyrosyl radical at the active site seems likely. Subsequent steps can be effectively blocked by mutation of a residue at the surface of the protein, apparently preventing the entry of protons.

Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I.

Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis. The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer. There are 20 conserved residues in subunit I and two in subunit II. Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E. coli. This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively. The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane. The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices. The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d. There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E. coli cytochrome bd-I numbering). It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface. The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses. Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.

EPR studies of the cytochrome-d complex of Escherichia coli.

We have examined the thermodynamic and EPR properties of one of the ubiquinol oxidase systems (the cytochrome d complex) of Escherichia coli, and have assigned the EPR-detectable signals to the optically identified cytochromes. The axial high spin g = 6.0 signal has been assigned to cytochrome d based on the physicochemical properties of this signal and those of the optically defined cytochrome d. A rhombic low spin species at gx,y,z = 1.85, 2.3, 2.5 exhibited similar properties but was present at only one-fifth the concentration of the axial high spin species. Both species have an Em7 of 260 mV and follow a -60 mV/pH unit dependence from pH 6 to 10. The rhombic high spin signal with gy,z = 5.5 and 6.3 has been assigned to cytochrome b-595. This component has an Em7 of 136 mV and follows a -30 mV/pH unit dependence from pH 6 to 10. Lastly, the low spin gz = 3.3 signal which titrates with an Em7 of 195 mV and follows a -40 mV/pH unit dependence from pH 6 to 10 has been assigned to cytochrome b-558. Spin quantitation of the high-spin signals indicates that cytochrome d and b-595 are present in approximately equal amounts. These observations are discussed in terms of the stoichiometry of the prosthetic groups and its implications on the mechanism of electron transport.

Quasi-elastic light scattering studies on pyruvate oxidase.

Quasi-elastic or dynamic light scattering has been used to examine the translational diffusion properties of the enzyme pyruvate oxidase (pyruvate: ferricytochrome beta 1 oxidoreductase, EC 1.2.2.2.). Controlled proteolysis of the enzyme converts the native form of the enzyme to a protease-activated form which has a specific activity about 20-fold greater than the native oxidase. Light scattering studies indicate no significant change in the size or shape of pyruvate oxidase as a result of this proteolytic activation. In both cases the enzyme may be characterized as a hydrated sphere with a Stokes radius of about 53A. The sedimentation velocity-diffusion technique was used to obtain the molecular weight of this tetrameric enzyme, about 252 000 with a value of f/f0 of 1.25.

Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases.

A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.

Recent studies of the cytochrome o terminal oxidase complex of Escherichia coli.

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of Escherichia coli when the bacteria are grown under conditions of high aeration. The oxidase is a ubiquinol oxidase and reduces molecular oxygen to water. Electron transport through the enzyme is coupled to the generation of a protonmotive force. The purified cytochrome o complex contains four or five subunits, two protoheme IX (heme b) prosthetic groups, plus at least one Cu. The subunits are all encoded by the cyo operon. Sequence comparisons show that the cytochrome o complex is closely related to the aa3-type cytochrome c oxidase family. Gene fusions have been used to define the topology of each of the gene products. Subunits I, II, III and IV are proposed to have 15, 2, 5 and 3 transmembrane spans, respectively. The fifth gene product (cyoE) encodes a protein with 7 membrane spanning segments, and this may also be a subunit of this enzyme. Fourier transform infrared spectroscopy has been used to monitor CO bound in the active site where oxygen is reduced. These data provide definitive proof that the cytochrome o complex has a heme-copper binuclear center, similar to that present in the aa3-type cytochrome c oxidases. Site-directed mutagenesis is being utilized to define which amino acids are ligands to the heme iron and copper prosthetic groups.

One-step purification of histidine-tagged cytochrome bo3 from Escherichia coli and demonstration that associated quinone is not required for the structural integrity of the oxidase.

The cytochrome bo3 ubiquinol oxidase from Escherichia coli is a member of the heme-copper superfamily of proton-pumping respiratory oxidases. An improved preparative protocol was desired that would minimize the potential damage during protein isolation of labile mutants of the oxidase. Variants of the oxidase containing a histidine tag at the carboxy-terminus of either subunit I, II or III were constructed. The constructs with the histidine tag on either subunit I or II successfully allowed the enzyme to be isolated with high purity in one step using Ni2+ affinity chromatography. The enzyme with the histidine tag on subunit II is particularly useful insofar as the enzyme isolated in this manner has little, if any, heterogeneity resulting from the presence of heme O in the low spin heme-binding site, i.e., cytochrome oo3 is minimized. The enzyme can be prepared in virtually any quantity very rapidly and is suitable for biophysical characterization. Cytochrome bo3 was prepared in either Triton X-100, sucrose monolaurate, or dodecyl maltoside. The enzyme isolated in the presence of either sucrose monolaurate or dodecyl maltoside contains approximately one equivalent of associated ubiquinone, whereas this is absent when Triton X-100 is used. However, the UV/vis absorbance and steady-state kinetic properties of the enzyme are virtually identical regardless of which detergent is used. These data are consistent with previous reports that cytochrome bo3 contains an equivalent of 'tightly associated' ubiquinone, but clearly demonstrate that this quinone can be removed without damaging the enzyme and is not critical to the maintenance of the native structure of the oxidase.

Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations.

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3. This suggests that the binuclear centers of the two enzymes are quite similar.

Studies on the complex formed between bacitracin A and divalent cations.

Bacitracin A is a peptide antibiotic which forms stoichiometric complexes with divalent cations, including Ni2+ and Zn2+. In this paper it is shown that the metal-bacitracin complex contains a group which has a pKa near pH 5.5. Deprotonation of the group is concomitant with the aggregation and precipitation of the metal-bacitracin complex. Bacitracin A, in the absence of metals, does not contain any group which has a pKa in this range. It is postulated that this group is the N-terminal amino of isoleucine, which was previously postulated not to be directly involved in metal coordination based on proton release measurements. An attempt was made to demonstrate directly that the N-terminal amino group is not coordinated to the metal by examining the reactivity of this group with 2,4,6-trinitrobenzene sulfonate. It was clearly shown that bound metals protect the N-terminal amino group from reacting with this reagent. It is speculated that this metal-protection results from a combination of factors, including steric hindrance.

Conformational studies of Escherichia coli pyruvate oxidase.

Pyruvate oxidase (pyruvate:oxygen oxidoreductase (phosphorylating), EC 1.2.3.3) is a peripheral membrane enzyme from Escherichia coli which utilizes the cofactors thiamin pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) to catalyze the decarboxylation of pyruvate to acetic acid and carbon dioxide. The specific activity of the oxidase is enhanced 25-fold when assayed in the presence of certain lipids and detergents. Previous studies have demonstrated that the affinity of pyruvate oxidase for phospholipids and detergents is substantially increased when the flavin is reduced. In this paper, several techniques are utilized to probe both the nature of the active site and the conformational changes in the protein which are concomitant with flavin reduction and with the binding of lipids to the enzyme. Analysis of the circular dichroism spectrum in the far ultraviolet region indicates that neither the binding of lipid activators to the oxidase nor reduction of the enzyme-bound flavin by pyruvate has a significant effect on the average secondary structure of the enzyme. High-resolution electron microscopy demonstrates that at low enzyme concentrations, i.e., assay conditions, incubation of the reduced flavoprotein in the presence of an amphiphilic activator does not alter the quaternary structure of pyruvate oxidase. The results indicate that the conformational changes in the protein due either to reduction of the flavin or to the binding of lipid activators are localized.

Relationships between membrane-bound cytochrome o from Vitreoscilla and that of Escherichia coli.

The cytochrome o terminal oxidases from the bacteria Vitreoscilla and Escherichia coli are structurally and functionally related. They have similar optical spectra, both exhibit ubiquinol-1 oxidase activity and are inhibited similarly. Both enzymes contain four subunits by SDS-polyacrylamide gel electrophoresis analysis and contain protoheme IX and Cu2+ prosthetic groups. Antibodies raised against the oxidase purified from E. coli crossreact with the Vitreoscilla oxidase.