Ultrasonography in preclinical education: a pilot study.

Context: Ultrasonography is a valuable diagnostic tool in the clinical setting. Yet, medical students often have minimal familiarity with this technology.

Objective: To evaluate the ability of second-year medical students to use ultrasonography for identification of anatomic structures and pathologic conditions.

Roles of the ccoGHIS gene products in the biogenesis of the cbb(3)-type cytochrome c oxidase.

In many bacteria the ccoGHIS cluster, located immediately downstream of the structural genes (ccoNOQP) of cytochrome cbb(3) oxidase, is required for the biogenesis of this enzyme. Genetic analysis of ccoGHIS in Rhodobacter capsulatus demonstrated that ccoG, ccoH, ccoI and ccoS are expressed independently of each other, and do not form a simple operon. Absence of CcoG, which has putative (4Fe-4S) cluster binding motifs, does not significantly affect cytochrome cbb(3) oxidase activity.

Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase.

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P.

Cu XAS shows a change in the ligation of CuB upon reduction of cytochrome bo3 from Escherichia coli.

Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane.

FT-IR analysis of membranes of Rhodobacter sphaeroides 2.4.3 grown under microaerobic and denitrifying conditions.

Fourier transform infrared spectroscopic analysis of CO binding proteins in Rhodobacter sphaeroides reveals the presence of a membrane-bound nitric oxide reductase (Nor). Nor has been clearly distinguished from the cytochrome oxidases by the temperature-dependence of relaxation following photodissociation of the CO complex at cryogenic temperatures. The center frequency and band shape, 1970 cm-1 and 20-30 cm-1 width at half-peak height, are similar to those reported for resonance Raman spectra of purified Paracoccus denitrificans Nor.

Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.

Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways.

Polar residues in helix VIII of subunit I of cytochrome c oxidase influence the activity and the structure of the active site.

The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site-directed mutants identified the ligands of heme a, heme a3, and CuB [Hosler et al. (1993) J.

Photoperturbation of the heme a3-CuB binuclear center of cytochrome c oxidase CO complex observed by Fourier transform infrared spectroscopy.

Purified cytochrome c oxidase CO complex from beef heart has been studied by Fourier transform infrared absorbance difference spectroscopy. Photolysis at 10-20 Kelvin results in dissociation of a3FeCO, formation of CuBCO, and perturbation of the a3-heme and CuB complex. The vibrational perturbation spectrum between 900 and 1700 cm-1 contains a wealth of information about the binuclear center.

A pH-dependent polarity change at the binuclear center of reduced cytochrome c oxidase detected by FTIR difference spectroscopy of the CO adduct.

A pH-dependent polarity change at the heme-copper binuclear center of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been identified by low-temperature FTIR difference spectroscopy. "Light"-minus-"dark" FTIR difference spectra of the fully reduced CO-enzyme adduct were recorded at a range of pH, and the dominance of different populations of bound CO, alpha and beta, was found to vary with pH. An apparent pKa of about 7.

A ligand-exchange mechanism of proton pumping involving tyrosine-422 of subunit I of cytochrome oxidase is ruled out.

The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system.

The highly conserved methionine of subunit I of the heme-copper oxidases is not at the heme-copper dinuclear center: mutagenesis of M110 in subunit I of cytochrome bo3-type ubiquinol oxidase from Escherichia coli.

A common feature within the heme-copper oxidase superfamily is the dinuclear heme-copper center. Analysis via extended X-ray absorption fine structure (EXAFS) has led to the proposal that sulfur may be bound to CuB, a component of the dinuclear center, and a highly conserved methionine (M110 in the E. coli oxidase) in subunit I has been proposed as the ligand.

Site-directed mutagenesis of residues within helix VI in subunit I of the cytochrome bo3 ubiquinol oxidase from Escherichia coli suggests that tyrosine 288 may be a CuB ligand.

The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxidases, as well as most prokaryotic respiratory oxidases. All members of the superfamily have a subunit homologous to subunit I of the mammalian cytochrome c oxidases. This subunit provides the amino acid ligands to a low-spin heme component as well as to a heme-copper binuclear center, which is the site where dioxygen is reduced to water.

A novel cytochrome c oxidase from Rhodobacter sphaeroides that lacks CuA.

Rhodobacter sphaeroides contains at least two different cytochrome c oxidases. When these bacteria are grown with high aeration, the traditional aa3-type cytochrome c oxidase is present at relatively high levels. However, under microaerophilic growth conditions or when the bacteria are grown photosynthetically, the amount of the aa3-type oxidase is greatly diminished and an alternate cytochrome c oxidase is evident.

A loop between transmembrane helices IX and X of subunit I of cytochrome c oxidase caps the heme a-heme a3-CuB center.

Site-directed mutants were prepared of four consecutive and highly conserved residues (His-411, Asp-412, Thr-413, Tyr-414) of an extramembrane loop that connects putative transmembrane helices IX and X of subunit I of Rhodobacter sphaeroides cytochrome c oxidase. The modified enzymes were purified and analyzed by optical, resonance Raman, FTIR, and EPR spectroscopies. Consistent with our recent model in which both hemes are ligated to histidines of helix X [Hosler, J.

Spectroscopic characterization of mutants supports the assignment of histidine-419 as the axial ligand of heme o in the binuclear center of the cytochrome bo ubiquinol oxidase from Escherichia coli.

The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of heme-copper oxidases which also includes the aa3-type cytochrome c oxidases. The oxygen-binding binuclear center of cytochrome bo is located in subunit I and consists of a heme (heme o; heme a3 in the aa3-type oxidases) and a copper (Cu(B)). Previous spectroscopic studies have shown that heme o is bound to the protein via a single histidine residue.

Site-directed mutants of the cytochrome bo ubiquinol oxidase of Escherichia coli: amino acid substitutions for two histidines that are putative CuB ligands.

The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases. The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme. The high-spin heme is magnetically coupled to a copper, CuB, forming a binuclear center which is the site of oxygen reduction to water.

Site-directed mutagenesis of highly conserved residues in helix VIII of subunit I of the cytochrome bo ubiquinol oxidase from Escherichia coli: an amphipathic transmembrane helix that may be important in conveying protons to the binuclear center.

Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily of heme-copper respiratory oxidases. This superfamily, which includes the eukaryotic cytochrome c oxidases, has in common a bimetallic center consisting of a high-spin heme component and a copper atom (CuB) which is the site where molecular oxygen is reduced to water. Subunit I, which contains all the amino acid ligands to the metal components of the binuclear center, has 15 putative transmembrane spanning helices, of which 12 are common to the entire superfamily.

Substitution of asparagine for aspartate-135 in subunit I of the cytochrome bo ubiquinol oxidase of Escherichia coli eliminates proton-pumping activity.

The terminal quinol oxidase, cytochrome bo, of Escherichia coli is a member of the large terminal oxidase family, which includes cytochrome aa3-type terminal oxidases from bacteria, plants, and animals. These enzymes conserve energy by linking electron transfer to vectorial proton translocation across mitochondrial or bacterial cell membranes. Site-directed mutagenesis of the five most highly conserved acidic amino acids in subunit I of cytochrome bo was performed to study their role in proton transfer.

Identity of the axial ligand of the high-spin heme in cytochrome oxidase: spectroscopic characterization of mutants in the bo-type oxidase of Escherichia coli and the aa3-type oxidase of Rhodobacter sphaeroides.

Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases. These enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms. Two such heme-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli.

Spectroscopic evidence for a heme-heme binuclear center in the cytochrome bd ubiquinol oxidase from Escherichia coli.

The cytochrome bd complex is a ubiquinol oxidase, which is part of the aerobic respiratory chain of Escherichia coli. This enzyme is structurally unrelated to the heme-Cu oxidases such as cytochrome c oxidase. While the cytochrome bd complex contains no copper, it does have three heme prosthetic groups: heme b558, heme b595, and heme d (a chlorin).

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.

Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga.

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.

Structural perturbation of the a3-CuB site in mitochondrial cytochrome c oxidase by alcohol solvents.

Ethanol has been observed to cause a perturbation of the catalytic center of the major respiratory protein cytochrome c oxidase. These effects were examined by Fourier transform infrared spectroscopy of carbon monoxide complexes of cytochrome a3Fe and of CuB formed by low-temperature photodissociation of the a3FeCO complex. Carbon monoxide binds to reduced cytochrome oxidase in two major structural forms, alpha and beta, both of which are altered by ethanol.