Altered Blood Flow in the Ophthalmic and Internal Carotid Arteries in Patients with Age-Related Macular Degeneration Measured Using Noncontrast MR Angiography at 7T.

BACKGROUND AND PURPOSE: Age-related macular degeneration is associated with reduced perfusion of the eye; however, the role of altered blood flow in the upstream ophthalmic or internal carotid arteries is unclear. We used ultra-high-field MR imaging to investigate whether the diameter of and blood flow in the ophthalmic artery and/or the ICA are altered in age-related macular degeneration and whether any blood flow changes are associated with disease progression. MATERIALS AND METHODS: Twenty-four patients with age-related macular degeneration and 13 similarly-aged healthy controls participated. TOF and high-resolution dynamic 2D phase-contrast MRA (0.26 × 0.26 × 2mm3, 100-ms effective sampling rate) was acquired at 7T. Vessel diameters were calculated from cross-sectional areas in phase-contrast acquisitions. Blood flow time-series were measured across the cardiac cycle. RESULTS: The ophthalmic artery vessel diameter was found to be significantly smaller in patients with age-related macular degeneration than in controls. Volumetric flow through the ophthalmic artery was significantly lower in patients with late age-related macular degeneration, with a significant trend of decreasing volumetric ophthalmic artery flow rates with increasing disease severity. The resistance index was significantly greater in patients with age-related macular degeneration than in controls in the ophthalmic artery. Flow velocity through the ophthalmic artery and ICA was significantly higher in patients with age-related macular degeneration. Ophthalmic artery blood flow as a percentage of ipsilateral ICA blood flow was nearly double in controls than in patients with age-related macular degeneration. CONCLUSIONS: These findings support the hypothesis that vascular changes upstream to the eye are associated with the severity of age-related macular degeneration. Additional investigation into the potential causality of this relationship and whether treatments that improve ocular circulation slow disease progression is warranted.

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. Recent models of subunit I, however, suggest that this residue is unlikely to be near CuB, but is predicted to be near the low spin heme component of the heme-copper oxidases. In this paper, the role of M110 is examined by spectroscopic analyses of site-directed mutants of the bo3-type oxidase from Escherichia coli. The results show that M110 is a non-essential residue and suggest that it is probably not near the heme-copper dinuclear center.

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. The amino acid sequence of transmembrane helix VI of subunit I is the most highly conserved within the superfamily. Previous efforts have demonstrated that one of the residues in this region, H284, is critical for oxidase activity and for the assembly of CuB. This paper presents the analysis of additional site-directed mutants in which other highly conserved residues in helix VI (P285, E286, Y288, and P293) have been substituted. Most of the mutants are enzymatically inactive. Structural perturbations reported by Fourier transform infrared absorption difference spectroscopy of CO adducts of the mutant oxidases confirm the previous suggestion that this region is adjactent to CuB. Furthermore, the analysis of five different substitutions for Y288 indicates that all lack CuB. On the basis of these data, it is proposed that Y288 may be a CuB ligand along with H333, H334, and H284, and a plausible molecular model of the CuB site is presented.

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.

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.

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 mutagenesis of conserved histidine residues in subunit I has identified two residues (H284 and H419) which are candidates for the ligand of heme o, while spectroscopic studies of mutants at H284 definitively demonstrated that this residue cannot be the axial ligand. Consequently, the single remaining conserved histidine in subunit I (H419) was assigned as the ligand for the heme of the binuclear center. In this paper, this assignment is tested by characterization of additional mutants in which the putative heme o axial ligand, H419, is replaced by other amino acids. All mutations at H419 result in the loss of enzyme activity. Analyses via UV-visible and Fourier transform infrared spectroscopies reveal that substantial perturbation has occurred at the binuclear center as a result of the amino acid substitutions. In contrast with the wild-type enzyme, the mutant enzymes bind very little carbon monoxide. Three other amino acid residues which are potential ligands for heme o are shown tob e nonessential for enzyme activity. Mutations in these residues do not perturb the UV-visible or FTIR spectroscopic characteristics of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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. Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases. It has been proposed previously that the two adjacent histidines in putative transmembrane helix VII (H333 and H334 in the E. coli sequence) of the largest subunit of the heme-copper oxidases are ligands to CuB. Previously reported mutagenesis studies of the E. coli bo-type oxidase and the aa3-type oxidase of Rhodobacter sphaeroides supported this model, as substitutions at these two positions produced nonfunctional enzymes but did not perturb the visible spectra of the two heme groups. In this work, six different amino acids, including potential copper-liganding residues, were substituted for H333 and H334 of the E. coli oxidase. All of the mutations resulted in inactive, but assembled, oxidase with both of the heme components present. However, cryogenic Fourier transform infrared (FTIR) spectroscopy of the CO adducts revealed that dramatic changes occur at the binuclear center as a result of each mutation and that CuB appears to be absent.(ABSTRACT TRUNCATED AT 250 WORDS)

The gateway to the active site of heme-copper oxidases.

The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases. The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels. CO recombination to the wild-type ubiquinol oxidase exhibited saturation kinetics (kmax = 190 s-1, Km = 2.4 mM), indicative of the existence of an intermediate in the ligand-binding pathway. FTIR spectroscopy and TRIR spectroscopy were used to demonstrate conclusively that this intermediate was a CuB-CO complex. Two mutant oxidases (His333Leu, His334Leu) which lack CuB showed no evidence of saturation of CO rebinding, even up to 21 mM CO. Also, the absolute rates of CO binding to the mutant oxidases were much greater than for wild type, even at CO concentrations well below the apparent Km for wild-type enzyme. These results clearly indicate that the copper ion at the binuclear site acts as an obligatory way station, or gate, severely limiting the approach of ligands to the heme active site. Further, an analysis of the rate constants for CO binding to CuB suggests that the protein structure external to the binuclear site regulates ligand entry into this site. We propose that these control mechanisms for substrate binding are operative throughout this general class of enzymes.

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. These enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center. Energy conservation is accomplished by coupling electron transfer through the metals of the oxidases to proton translocation across the cellular membrane. The Rb. sphaeroides and E. coli enzymes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a), as well as additional histidine residues which are probable ligands for copper (CuB). However, the histidine that binds the heme of the binuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419). Additional characterization by Fourier transform infrared spectroscopy of the CO-bound forms of the E. coli enzyme in which His284 is replaced by glycine or leucine demonstrates that these mutations cause only subtle changes to CO bound to the heme of the binuclear center. Resonance Raman spectroscopy of the Rb. sphaeroides enzyme in which His284 is replaced by alanine shows that the iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419. These results demonstrate that His284 is not the heme a3 ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial NADH-quinone oxidoreductases: iron-sulfur clusters and related problems.

Many bacteria contain proton-translocating membrane-bound NADH-quinone oxidoreductases (NDH-1), which demonstrate significant genetic, spectral, and kinetic similarity with their mitochondrial counterparts. This review is devoted to the comparative aspects of the iron-sulfur cluster composition of NDH-1 from the most well-studied bacterial systems to date.: Paracoccus denitrificans, Rhodobacter sphaeroides, Escherichia coli, and Thermus thermophilus. These bacterial systems provide useful models for the study of coupling Site I and contain all the essential parts of the electron-transfer and proton-translocating machinery of their eukaryotic counterparts.

Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli.

The nature of the Escherichia coli membrane-bound NADH dehydrogenases and their role in the generation of the proton motive force has been controversial. One E. coli NADH:ubiquinone oxidoreductase has previously been purified to homogeneity, and its corresponding gene (ndh) has been isolated. However, two biochemically distinct E. coli NADH:ubiquinone oxidoreductase activities have been identified by others (K. Matsushita, T. Ohnishi, and H. R. Kaback, Biochemistry 26:7732-7737, 1987). An insertional mutation in the ndh gene has been introduced into the E. coli chromosome, and the resulting strain maintains membrane-bound NADH dehydrogenase activity, demonstrating that a second genetically distinct NADH dehydrogenase must be present. By standard genetic mapping techniques, the map position of a second locus (nuo) involved in the oxidation of NADH has been determined. The enzyme encoded by this locus probably translocates protons across the inner membrane, contributing to the proton motive force.

Energetic efficiency of Escherichia coli: effects of mutations in components of the aerobic respiratory chain.

The aerobic respiratory chain of Escherichia coli can function with either of two different membrane-bound NADH dehydrogenases (NDH-1 and NDH-2) and with either of two ubiquinol oxidases (bd-type and bo-type). The amounts of each of these enzymes present in the E. coli membrane depend on growth conditions in general and particularly on the dissolved oxygen concentration. Previous in vitro studies have established that NDH-1 and NDH-2 differ in the extent to which they are coupled to the generation of an energy-conserving proton motive force. The same is true for the two ubiquinol oxidases. Hence, the bioenergetic efficiency of the aerobic respiratory chain must depend on the electron flux through each of the specific enzyme components which are being utilized. In this work, the specific rates of oxygen consumption for cells growing under glucose-limited conditions are reported for a series of isogenic strains in which one or more respiratory components are genetically eliminated. The results are compatible with the proton translocation values of the various components reported from in vitro measurements. The data show that (i) the bd-type oxidase is less efficient than is the bo-type oxidase, but the former is still a coupling site in the respiratory chain; and (ii) under the conditions employed, the wild-type strain uses both the NDH-1 and NDH-2 NADH dehydrogenases to a significant degree, but most of the electron flux is directed through the bo-type oxidase.

Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa3 and cytochrome bo.

Cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo of E. coli are useful models of the more complex cytochrome c oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the heme a3 (or o)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.

The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a 'g = 12' EPR feature analogous to that of 'slow' cytochrome oxidase.

The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa3 type cytochrome oxidase in subunit I, which contains the catalytic core. The cytochrome bo enzyme forms a formate complex which exhibits 'g = 12' and 'g = 2.9' EPR signals at X band; similar signals have previously been observed only in association with the 'slow' and formate-ligand states of cytochrome oxidase. These signals arise from transitions within integral spin multiples identified with the homologous heme-copper binuclear catalytic centers in both enzymes.

Determination of the ligands of the low spin heme of the cytochrome o ubiquinol oxidase complex using site-directed mutagenesis.

The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant respiratory terminal oxidase when the bacteria are grown under high oxygen tension. The amino acid sequences of three of the subunits of this quinol oxidase reveal a substantial relationship to the aa3-type cytochrome c oxidases. The two cytochrome components (b563.5 and o) and the single copper (CuB) present in the E. coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome a3, and CuB of the aa3-type cytochrome c oxidases, respectively. These three prosthetic groups are all located within subunit I of the oxidase. Sequence alignments indicate only six totally conserved histidine residues among all known sequences of subunit I of the cytochrome c oxidases of various species plus the E. coli quinol oxidase. Site-directed mutagenesis has been used to change each of these totally conserved histidines with the presumption that two of these six must ligate to the low spin cytochrome center of the E. coli oxidase. The presence of the low spin cytochrome b563.5 component of the oxidase can be evaluated both by visible absorbance properties and by its EPR spectrum. The results unambiguously indicate that His-106 and His-421 are the ligands of the six-coordinate low spin cytochrome b563.5. Although the data are not definitive in making additional metal ligation assignments of the remaining four totally conserved histidines, a reasonable model is suggested for the structure of the catalytic core of the cytochrome o complex and, by extrapolation, of cytochrome c oxidase.