Laura Lemieux | University of Waterloo, Canada (original) (raw)

Papers by Laura Lemieux

Biochemistry, 1993

The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of hemecopper ox... more The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of hemecopper 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 0; heme a3 in the aa3-type oxidases) and a copper (CUB). 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 0, 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 hemeo are shown to be nonessential for enzyme activity. Mutations in these residues do not perturb the UV-visible or FTIR spectroscopic characteristics of the enzyme. These results are consistent with the assignment of H419 as the axial ligand of heme o in the E. coli enzyme and, by analogy, heme a3 in the aa3-type cytochrome c oxidases. The cytochrome bo quinol oxidase from Escherichia coli and the aa3-type cytochrome c oxidases are members of a superfamily of structurally related heme-copper respiratory oxidases (Chepuri et al., 1990b; Saraste et al., 1989). The heme-copper binuclear center, the site of oxygen reduction to water, is diagnostic of this class of respiratory oxidases, and consists of a high-spin heme magnetically coupled to a copper, denoted CUB (Chepuri et al., 1990a; Hill et al., 1992; Hosler et al., 1993; Woodruff, 1993). A second heme, which is sixcoordinate and low-spin, is also present in all members of this superfamily. Vectorial proton translocation across the membrane bilayer appears to be another common feature of the heme-copper oxidases (

Biochimica Et Biophysica Acta - Bioenergetics, Jul 1, 1990

The cytochrome o complex is the predominant terminal oxidase in the aerobic respiratory chain of ... more 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.

Journal of Biological Chemistry, 1992

The cytochrome o complex of Escherichia coli is a ubiquinol oxidase which is the predominant resp... more 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 and 0) and the single copper (CUB) present in the E. coli quinol oxidase appear to be equivalent to cytochrome a, cytochrome as, 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 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 bS63.6. 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.

Biochemical Society Transactions, Aug 1, 1991

Journal of Biological Chemistry, Jul 1, 1990

Biochemistry, Aug 3, 1993

The cytochrome bo ubiquinol oxidase from Escherichia coli is a five-subunit enzyme which is a mem... more The cytochrome bo ubiquinol oxidase from Escherichia coli is a five-subunit enzyme which is a member of the superfamily of heme-copper respiratory oxidases. Three of the subunits (I, II, and III) are homologous to the three mitochondrial encoded subunits of the eukaryotic aa3-type cytochrome c oxidase. Subunits, I, II, and III of the eukaryotic oxidase contain 12, 2, and 7 putative transmembrane spans, respectively. The hydropathy profiles of the subunits of most other members of this oxidase superfamily are consistent with these structures. However, subunit I from the E. coli oxidase contains 15 transmembrane spans, with one additional span at the N-terminus and two additional spans at the C-terminus in comparison to the eukaryotic oxidase. The additional transmembrane helix at the N-terminus predicts that the amino terminal residue should be on the periplasmic side of the membrane. By deleting the intergenic region between the cyoA and cyoB genes, an in-frame fusion between subunit II (cyoA) and subunit I (cyoB) was generated. This links the C-terminus of subunit II, known to be on the periplasmic side of the membrane, to the N-terminus of subunit I. The resulting oxidase is fully active, and supports the toplogical folding pattern previously suggested for subunit I with the N-terminus in the periplasm. Whereas subunit I of the E. coli oxidase has two additional membrane-spanning helices at the C-terminus, subunit III has two fewer helices than does the corresponding subunit III of the eukaryotic oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, Nov 8, 1994

The heme-copper oxidase superfamily contains all of the mammalian mitochondrial cytochrome c oxid... more 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.

Annals of the New York Academy of Sciences, Dec 1, 1988

Biochemistry, Oct 1, 1993

Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily o... more Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily of hemecopper 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. Transmembrane helix VI11 has been noted to contain highly conserved polar residues that fall along one face of the helix. These residues could, in principle, be important components of a pathway providing a conduit for protons from the cytoplasm to gain access to the binuclear center. These conserved residues include Thr352, Thr359, and Lys362. In addition, Pro358, in the middle of this transmembrane helix, is totally conserved in the superfamily. Some substitutions for Thr352 (Ala, Asn) result in major perturbations at the binuclear center as judged by the low-temperature Fourier transform infrared (FTIR) absorbance difference spectroscopy of the CO adducts. Whereas Thr352Ala is inactive enzymatically, both Thr352Asn and Thr352Ser have substantial activity. Substitutions for Thr359 (Ala or Ser) also do not perturb the spectroscopic properties of the binuclear metal center, but the Thr359Ala mutant is devoid of enzyme activity. Changing the neighboring Pro358 to Ala has no detectable effect on 7 Supported by US. Department of Energy Grant DE-FG-02-87ER13716 (to R.B.G.) and by National Science Foundation Grant DMB89-04614 (to J.O.A.).

The cytochrome o ubiquinol oxidase is one of two terminal oxidases present in the aerobic respira... more The cytochrome o ubiquinol oxidase is one of two terminal oxidases present in the aerobic respiratory chain of E. coli. The cytochrome o complex has been purified and found to contain two protoheme IXs and one copper atom. Subsequently the gene encoding the cyo operon has been cloned. The research presented in this thesis focuses on the continued structural analysis of the cytochrome o oxidase complex including the following areas of interest: (i) the DNA sequence of the cyo operon, (ii) subunit analysis, (iii) localization of the prosthetic groups of this complex to specific subunits and (iv) determination of the histidines responsible for ligating the prosthetic groups of this complex.The cyo DNA sequence presented here reveals that there are five open reading frames, cyo A, B, C, D, and E. Similarity searches performed on these putative subunits reveal that three of these ORFs are related to subunits II, I, and III of both bacterial and eukaryotic cytochrome c oxidases. The remaining two putative subunits have sequence similarity with subunits found in bacterial cytochrome c oxidases.Immunological analyses of subclones of cyoA and cyoB demonstrate that they encode subunits II and I, respectively. Subsequent spectroscopic analysis of these subclones and the cyoBCDE subclone localize both the high and low-spin hemes associated with this complex to subunit I (cyoB) of the cytochrome o oxidase.The alignment of subunit I from cytochrome o oxidase with the analogous subunit from over 23 different species of bacterial and eukaryotic aa$\sb3$-type cytochrome c oxidases revealed that there are seven conserved histidines referred to as H106, H284, H333, H334, H411, H419 and H421. Physical data on leucine mutations of these histidines indicate that H106 and H421 serve as ligands for the low-spin heme, H333 and H334 are likely to be copper ligands and H284 is probably the high-spin heme ligand.U of I OnlyETDs are only available to UIUC Users without author permissio

Biochemistry, Dec 1, 1993

FEBS Letters, Jul 24, 1995

A common feature within the heine-copper oxidase superfamily is the dinuclear heine-copper center... more A common feature within the heine-copper oxidase superfamily is the dinuclear heine-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 (Mll0 in the E. coil oxidase) in snbanit 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 heine component of the heine-copper oxidases. In this paper, the role of Mll0 is examined by spectroscopic analyses of sitedirected mutants of the bo~-type oxidase from Escherichia coil The results show that Mll0 is a non-essential residue and suggest that it is probably not near the heine-copper dinuclear center. Key words." ???? subunit I of the heme-copper oxidases. This methionine, which is present in all but three species, has been proposed as a ligand to Cu B in the binuclear center [5]. However, this is incompatible with recent models of subunit I based on site-directed mutagenesis studies of two bacterial heme-copper oxidases, the bo3-type oxidase from E. coli and aa3-type oxidase from Rhodobacter sphaeroides [4,7]. In these models, MII0 is located four residues below the histidine (H106) identified as a ligand for the low spin heine b component of the E. coli oxidase [8,9], which corresponds to the heme a component of the aa3-type oxidases [4,7,10,11]. In this work, the role of Mll0 in the bo3-type oxidase from E. coli was examined by site-directed mutagenesis. The results clearly show that Mll0 is not an essential residue and is unlikely to be near the dinuclear center, consistent with the current models of subunit I of the hemecopper oxidases [4,7,10].

The EMBO Journal, 1992

The cupredoxin fold, a Greek key fl-barrel, is a common structural motif in a family of small blu... more The cupredoxin fold, a Greek key fl-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CUA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CUA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CUA site has been introduced, are similar to the EPR spectra of the native CUA site in cytochrome oxidase. These results give the first experimental evidence that CUA is bound to the subunit H of cytochrome c oxidase and open a new way to study this peculiar copper site.

Journal of Biological Chemistry, 1990

Biochemistry Usa, May 1, 1995

The oxygen reaction of wild-type and helix VI11 mutants of cytochrome bo3 from Escherichia coli, ... more The oxygen reaction of wild-type and helix VI11 mutants of cytochrome bo3 from Escherichia coli, and the associated proton uptake during this reaction, has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. We have focused on mutations in the transmembrane helix VI11 where protonatable residues have been exchanged, and mainly on the inactive mutants (Le., T352A, T359A, and K362L,-M, and-Q). The kinetics for electron transfer during oxidation

Biochemistry Usa, 1993

Biochemical Society transactions, 1991

Nature Medicine, 1995

Amyloid is a term for extracellular protein fibril deposits that have characteristic tinctorial a... more Amyloid is a term for extracellular protein fibril deposits that have characteristic tinctorial and structural properties. Heparan sulphate, or the heparan sulphate proteoglycan perlecan, has been identified in all amyloids and implicated in the earliest stages of inflammation-associated (AA) amyloid induction. Heparan sulphate interacts with the AA amyloid precursor and the beta-peptide of Alzheimer's amyloid, imparting characteristic secondary and tertiary amyloid structural features. These observations suggest that molecules that interfere with this interaction may prevent or arrest amyloidogenesis. We synthesized low-molecular-weight (135-1,000) anionic sulphonate or sulphate compounds. When administered orally, these compounds substantially reduced murine splenic AA amyloid progression. They also interfered with heparan sulphate-stimulated beta-peptide fibril aggregation in vitro.