Biomedical subjects

T Haltia

Publications and source records attributed to T Haltia.

11 recordsLinked to original sources

PubMed1992

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.

Copper↗

PubMed1991

Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases.

The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103]. It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.

Bacillus subtilis↗

PubMed1991

Properties of the two terminal oxidases of Escherichia coli.

Proton translocation coupled to oxidation of ubiquinol by O2 was studied in spheroplasts of two mutant strains of Escherichia coli, one of which expresses cytochrome d, but not cytochrome bo, and the other expressing only the latter. O2 pulse experiments revealed that cytochrome d catalyzes separation of the protons and electrons of ubiquinol oxidation but is not a proton pump. In contrast, cytochrome bo functions as a proton pump in addition to separating the charges of quinol oxidation. E. coli membranes and isolated cytochrome bo lack the CuA center typical of cytochrome c oxidase, and the isolated enzyme contains only 1Cu/2Fe. Optical spectra indicate that high-spin heme o contributes less than 10% to the reduced minus oxidized 560-nm band of the enzyme. Pyridine hemochrome spectra suggest that the hemes of cytochrome bo are not protohemes. Proteoliposomes with cytochrome bo exhibited good respiratory control, but H+/e- during quinol oxidation was only 0.3-0.7. This was attributed to an "inside out" orientation of a significant fraction of the enzyme. Possible metabolic benefits of expressing both cytochromes bo and d in E. coli are discussed.

Cardiolipins↗

PubMed1991

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.

Amino Acid Sequence↗

PubMed1991

Genes coding for cytochrome c oxidase in Paracoccus denitrificans.

Several loci on the Paracoccus denitrificans chromosome are involved in the synthesis of cytochrome c oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochrome c550 gene. An alignment of six promoter regions reveals only short common sequences.

Amino Acid Sequence↗

PubMed1990

A novel combination of prosthetic groups in a fungal laccase; PQQ and two copper atoms.

Extracellular laccase (benzenediol: oxygen oxidoreductase EC 1.10.3.2) from the lignin-degrading fungus, Phlebia radiata, was shown to contain a novel combination of electron carriers as its prosthetic groups. In addition to two copper atoms per enzyme molecule, one molecule of PQQ was included as a cofactor. The EPR spectrum exhibits features of type 1 and type 2 copper atoms. In the enzymatic reaction 4 molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. During the reaction coniferyl alcohol is transformed to dilignols.

Copper↗

PubMed1989

Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.

Chromatography, DEAE-Cellulose↗

PubMed1988

The Paracoccus denitrificans cytochrome aa3 has a third subunit.

The presence of a third polypeptide subunit in Paracoccus cytochrome c oxidase is demonstrated. This protein (apparent molecular mass 23 kDa) binds dicyclohexylcarbodiimide in membranes of aerobically grown bacteria and in the purified enzyme. The N-terminal amino-acid sequence of this dicyclohexylcarbodiimide-binding protein is identical to the deduced sequence of the COIII gene product [Raitio et al. (1987) EMBO J. 6, 2825-2833]. We conclude that the aa3-type oxidase in Paracoccus is composed of at least three subunits, which correspond to the three mitochondrially coded polypeptides in the eukaryotic enzyme.

Amino Acid Sequence↗

PubMed1980

Juvenile metachromatic leukodystrophy. Clinical, biochemical, and neuropathologic studies in nine new cases.

We describe nine patients with metachromatic leukodystrophy. Seven patients had the juvenile form; in two others, the age at onset was 1 year, but the clinical course was different from the late infantile form. The age at onset ranged from 1 to 18 years; the duration ranged from three to 17 years. Mental retardation associated with motor impairment and pathological EEG and electromyographic findings were the main clinical findings. In patients with early onset, mental retardation was almost the only symptom for the first ten years. Segmental demyelination, remyelination, onion bulb formation, and occasional perivascular macrophages containing metachromatic lipid were the main findings in sural nerves studied after biopsy. The mean arylsulfatase-A (ASA) activity was 1.3 nmoles of nitrocatechol sulfate per milligram of protein per 30 minutes in peripheral leukocytes of the patients, 62.0 in the heterozygotes, and 139.0 in the controls. The ASA band could not be detected in enzyme electrophoresis.

Adolescent↗

PubMed1979

Arylsulphatase A and B in juvenile metachromatic leukodystrophy.

A series of five living patients with juvenile metachromatic leukodystrophy (MLD), ten first-degree relatives, and a number of controls were subjected to biochemical investigations including quantitative determination of arylsulphatase A (ASA) and B (ASB) activities in peripheral leukocytes and polyacrylamide disc gel elctrophoresis of arylsulphatases. Five relatives were family members of four previously deceased patients with juvenile MLD. The mean ASA activity of the patients was 1.3 nmol of p-nitrocatechol sulphate hydrolysed in 30 min per mg protein. It was 84 nmol in the relatives, 129 nmol in other neurological patients and 136 nmol in normal controls. The corresponding ASB activity was 38 nmol in the patients, 49 nmol in the relatives, and 99 nmol in normal controls. An extremely low ASB activity, 3.4 nmol, was found in one relative. No ASA band could be visualised in the enzyme electrophoretic patterns of the patients' leukocytes but the bands representing ASB appeared normal. Seven relatives showed ASA bands weaker than normal, and the relative with low ASB activity exhibited very weak ASB band. The low ASB activity in the patients and heterozygotes may be a characteristic feature of the slowly progressive juvenile type MLD diagnosed in the present series.

Adult↗

PubMed1978

Recurrent multiple cranial neuropathies.

A report of 12 patients with recurrent multiple cranial neuropathy is presented. In most cases the palsies a accompanied a mild respiratory infection. Both motor and sensory cranial nerves were affected, the facial nerve and optic nerve being the most common. The symptoms were self-limited in course and steroid therapy seemed to hasten the recovery in the cases it was used. Only in a few cases a noticeable neurological deficiency remained. Aetiological aspects of the syndrome are discussed.

Adult↗