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M Lübben

Publications and source records attributed to M Lübben.

At least 19 recordsLinked to original sources
PubMed2000

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans.

A bacterioferritin was recently isolated from the anaerobic sulphate-reducing bacterium Desulfivibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841-6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron-coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate-reducing bacteria.

Anaerobiosis↗
PubMed2000

Multiple posttranslational modifications at distinct sites contribute to heterogeneity of the lipoprotein cytochrome bo(3).

The heme-copper cytochrome oxidase of Escherichia coli (cytochrome bo(3)) was tagged with oligohistidine at the C-terminus of the small noncatalytic subunit IV. After detergent solubilization, the enzyme was purified by a one-step procedure with immobilized metal affinity chromatography. Using different cytochrome bo(3) constructs as reference, the products were investigated by mass spectroscopical and immunological methods. Several posttranslational modifications of subunits II, III, and IV were observed: (1) N-terminal methionines of subunits III and IV are split off. (2) Fifty percent of subunit III polypeptides are acetylated, presumably at the N-terminal alanine. (3) Lipoprotein processing of subunit II involves cleavage of the signal peptide. (4) Maturation of subunit II [Ma, J., Katsonouri, A., and Gennis, R. B. (1997) Biochemistry 36, 11298-11303] alters the structure of the N-terminal cysteine by N-palmitoylation and S-glyceryldipalmitoylation. (5) A hexapeptide is split off from the C-terminus of subunit II. This happens subsequently to the N-terminal lipoprotein processing step and is dependent on the growth state of cells.

Amino Acid Sequence↗
PubMed1999

Extended heme promiscuity in the cyanobacterial cytochrome c oxidase: characterization of native complexes containing hemes A, O, and D, respectively.

The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 microliters packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase high-performance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a3 and o3, both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa3-type cytochrome c oxidase. The heme D, on the other hand, seems to form a spectrally distinguished, yet kinetically ill-defined hemoprotein complex which does not qualify as a fully functional d-type terminal oxidase on our (wild-type) cyanobacteria even after growth under semianaerobic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under dinitrogen-fixing conditions did not change this situation. Thus, we are left with (wild-type) cyanobacteria forming an unbranched respiratory chain with only a single type of terminal oxidase protein, viz. the known aa3-type cytochrome c oxidase. This oxidase, however, may incorporate different prosthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox environment. In particular, however, conditions for expression of the two quinol oxidases potentially and additionally coded for by the genome of, e. g., Synechocystis sp. PCC6803 (see http://www.kazusa.or.jp/cyano), have not yet been found.

Acids↗
PubMed1999

Electron transfer induces side-chain conformational changes of glutamate-286 from cytochrome bo3.

Heme-copper oxidases have two putative proton channels, the so-called K-channel and the membrane-spanning D-channel. The latter contains a number of polar groups with glutamate-286 located in its center, which could-together with bound water-contribute to a transmembrane hydrogen-bonded network. Protonation states of carboxyl groups from cytochrome bo3 of Escherichia coli were studied by redox Fourier transform infrared (FTIR) difference spectroscopy. A net absorbance increase in the carboxyl region was observed upon reduction. The band signature typically found in heme-copper oxidases comprises an absorbance decrease (reduced-minus-oxidized difference spectra) at 1745 cm-1 and increase at 1735 cm-1. No significant changes in the carboxyl region were found in the site-specific mutants D135E and D407N. The difference bands were lacking in redox spectra of mutants at position 286; they could clearly be related to Glu-286. In wild-type oxidase, the pK of Glu-286 appears to be higher than 9.8. Upon solvent isotope exchange from H2O to D2O, the band at 1745 cm-1 shifts more readily than the one at 1735 cm-1, indicating dissimilar accessibility of the carboxyl side chain to the hydrogen-bonded network in both redox states. The data are consistent with a redox-triggered conformational change of Glu-286, which attributes to the carboxyl group an orientation toward the interior of the D-channel for the oxidized form. The change of Glu-286 is retained in cyanide complexes of cytochrome bo3 and of cytochrome c oxidase; therefore it should be related to oxidoreduction of the heme b and/or CuB metal centers.

Aspartic Acid↗
PubMed1998

The active site of the bacterial nitric oxide reductase is a dinuclear iron center.

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.

Binding Sites↗
PubMed1998

Bacteriorhodopsin's intramolecular proton-release pathway consists of a hydrogen-bonded network.

In its proton-pumping photocycle, bacteriorhodopsin releases a proton to the extracellular surface at pH 7 in the transition from intermediate L to intermediate M. The proton-release group, named XH, was assigned in low-temperature FT-IR studies to a single residue, E204 [Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R. , and Lanyi, J. K. (1995) J. Biol. Chem. 270, 27122-27126]. The time-resolved room-temperature step-scan FT-IR photocycle studies on wild-type and E204Q-, and E204D-mutated bacteriorhodopsin, which we present here, show in contrast that the FT-IR data give no evidence for deprotonation of E204 in the L-to-M transition. Therefore, it is unlikely that E204 represents XH. On the other hand, IR continuum absorbance changes indicate intramolecular proton transfer via an H-bonded network to the surface of the protein. It appears that this H-bonded network is spanned between the Schiff base and the protein surface. The network consists at least partly of internally bound water molecules and is stabilized by E204 and R82. Other not yet identified groups may also contribute. At pH 5, the intramolecular proton transfer to the surface of the protein seems not to be disturbed. The proton seems to be buffered at the surface and later in the photocycle released into the bulk during BR recovery. Intramolecular proton transfer via a complex H-bonded network is proposed to be a general feature of proton transfer in proteins.

Bacteriorhodopsins↗
PubMed1996

The Bradyrhizobium japonicum coxWXYZ gene cluster encodes a bb3-type ubiquinol oxidase.

Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium, has a complex respiratory electron-transport chain, capable of functioning throughout a wide range of oxygen tensions. It does so by synthesizing a number of terminal oxidases, each appropriate for different environmental conditions. We have previously described the cloning of the large catalytic subunit, coxX, from one of the terminal oxidases from B. japonicum [Surpin, M.A., Moshiri, F., Murphy, A.M. and Maier, R.J. (1994) Genetic evidence for a fourth terminal oxidase from Bradyrhizobium japonicum. Gene 143, 73-77]. In this work, we describe the remaining subunits of this terminal oxidase complex, which is encoded by the coxWXYZ operon. The polypeptide encoded by coxW does not contain any amino acid residues that are known to bind the CuA atom of cytochrome c terminal oxidases, but contains residues thought to be involved in ubiquinol binding. Terminal oxidase cyanide inhibition titration pattern comparisons of the wild type with a coxWXYZ insertion mutant indicated the new oxidase is expressed microaerobically. However analysis of hemes extracted from microaerobically incubated cells revealed the absence of heme O in this strain (from both the wild type and the mutant) of B. japonicum. Therefore, coxWXYZ most likely encodes a microaerobically-expressed bb3-type ubiquinol oxidase.

Amino Acid Sequence↗
PubMed1996

Redox FTIR difference spectroscopy using caged electrons reveals contributions of carboxyl groups to the catalytic mechanism of haem-copper oxidases.

Redox spectra of the haem-copper oxidases cytochrome aa3 of Rhodobacter sphaeroides and cytochrome bo3 of Escherichia coli were recorded in the visible and infrared spectral regions. The reduction of oxidases was initiated after light activation of the 'caged electron' donor riboflavin. Infrared redox difference spectra exhibit absorbance changes in the amide I region, which are indicative of very small redox-linked conformational movements in the polypeptide backbone. A reproducible redox-dependent pattern of positive and negative absorption changes is found in the carbonyl region (1680-1750 cm(-1)). The carbonyl bands shift to lower frequencies due to isotope exchange of the solvent H2O to D2O. This common feature of cytochrome c and quinol oxidases indicates that at least (i) one redox-sensitive carboxyl group is in the protonated state in the oxidized form and (ii) one carboxylic acid is involved at a catalytic step--presumably in proton translocation--of haem-copper oxidase.

Catalysis↗
PubMed1995

New archaebacterial genes coding for redox proteins: implications for the evolution of aerobic metabolism.

Archaebacterial respiratory chains are poorly understood at the molecular level. We have cloned and sequenced a cluster of five new genes from the archaebacterium Sulfolobus acidocaldarius, four of them coding for redox proteins: a Rieske iron-sulphur protein, a cytochrome b, a subunit II of cytochrome oxidase and a blue copper protein (sulfocyanin). The fifth gene codes for a hydrophobic protein with no homologue in the databases. The gene organization and biochemical data suggest that all four redox proteins probably form part of a membrane respiratory complex together with SoxM, a previously characterized catalytic subunit of cytochrome oxidase. A phylogenetic analysis of the new protein sequences gives support to the view that an elaborate aerobic respiratory chain was already present in the last common ancestor of all living organisms.

Aerobiosis↗
PubMed1994

Novel prenylated hemes as cofactors of cytochrome oxidases. Archaea have modified hemes A and O.

A series of novel hemes with modifications of the isoprenyl side chain has been detected in archaea. Heme A(S) was isolated from cytochrome oxidases of the thermoacidophilic archaeon, Sulfolobus acidocaldarius. Heme A(S) has the same spectroscopic features as heme A but has a hydroxyethylgeranylgeranyl side chain instead of the hydroxyethylfarnesyl group. This variant is also present in other archaeal oxidases as well as in the cytochrome oxidases of a thermophilic eubacterium. Other archaea (Thermoplasma, Pyrobaculum) were also shown to have cytochrome oxidases. From these organisms, three novel prenylated heme variants (called OT, OP1, and OP2) were isolated. They are structurally related to heme O; OP2 has a hydroxyethylgeranylgeranyl instead of the hydroxyethylfarnesyl side chain. In OP1 and OT, the hydroxyethylprenyl group is altered to ethenylprenyl by elimination of a water molecule. Most probably, the novel hemes are cofactors binding to the binuclear reaction centers of archaeal cytochrome oxidases.

Archaea↗
PubMed1994

A second terminal oxidase in Sulfolobus acidocaldarius.

We previously found that the soxABCD operon encodes a quinol oxidase complex in Sulfolobus acidocaldarius and this enzyme was purified and characterized. In this study, we have used a cloning procedure based on the conservation of oxidase sequences and the polymerase chain reaction to isolate a new gene (soxM) encoding a subunit of another terminal oxidase. This terminal oxidase is a fusion between two central components of cytochrome oxidases, subunits I and III. soxM forms a transcriptional unit which is expressed under heterotrophic growth conditions. The corresponding protein was detected by direct protein sequencing in a preparation enriched with a cytochrome absorbing light at 562 nm. This preparation contains a terminal oxidase which is able to oxidize the artificial substrate N,N,N',N'-tetramethyl-p-phenylenediamine. This preparation also contains SoxC, a protein homologous to the mitochondrial cytochrome b, and a Rieske iron-sulphur center. We suggest that SoxM is the core component of a second terminal oxidase complex and that this complex may share a subunit (SoxC) with the SoxABCD complex.

Amino Acid Sequence↗
PubMed1994

Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.

Aerobiosis↗
PubMed1994

The terminal oxidases of Paracoccus denitrificans.

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding subunit I of the aa3-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (delta ctaDI, delta ctaDII), indicating that they are isoforms of subunit I of the aa3-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. This protohaem-containing oxidase, called cytochrome bb3, is the only quinol oxidase expressed under the conditions used. In a triple oxidase mutant (delta ctaDI, delta ctaDII, cyoB::KmR) an alternative cytochrome c oxidase has been characterized; this cbb3-type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

Aerobiosis↗
PubMed1994

The purified SoxABCD quinol oxidase complex of Sulfolobus acidocaldarius contains a novel haem.

A respiratory quinol oxidase complex that is encoded by the soxABCD operon has been purified from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The enzyme was solubilized with dodecyl maltoside and purified in the presence of this detergent and ethylene glycol. The complex is hydrodynamically homogeneous and contains at least five different polypeptides. In addition to the major subunits SoxA, SoxB and SoxC, it has two small polypeptides. One of these is the translation product of a short open reading frame (now called the soxD gene) at the end of the operon. The optical and electron paramagnetic resonance spectra of the SoxABCD complex have been characterized. It probably contains four A-type haems which are bound to SoxB and SoxC. The structure of these haems is not identical to haem A. The novel haem As has a 2-hydroxyethyl geranylgeranyl in position 2 of the porphyrin ring whereas haem A has the related farnesyl-containing side-chain.

Bacterial Proteins↗
PubMed1993

Bacillus subtilis CtaA and CtaB function in haem A biosynthesis.

Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem O is an intermediate. Possible roles of the Bacillus subtilis ctaA and ctaB gene products in haem O and haem A synthesis were studied. Escherichia coli does not contain haem A. The ctaA gene on plasmids in E. coli resulted in haem A accumulation in membranes. The presence of ctaB together with ctaA increased the amount of haem A found in E. coli. Haem O was not detected in wild-type B. subtilis strains. A previously isolated B. subtilis ctaA deletion mutant was found to contain haem B and haem O, but not haem A. B. subtilis ctaB deletion mutants were constructed and found to lack both haem A and haem O. The results with E. coli and B. subtilis strongly suggest that the B. subtilis CtaA protein functions in haem A synthesis. It is tentatively suggested that if functions in the oxygenation/oxidation of the methyl side group of carbon 8 of haem O. B. subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E. coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis.

Alkyl and Aryl Transferases↗
PubMed1992

An archaebacterial terminal oxidase combines core structures of two mitochondrial respiratory complexes.

The operon coding for a respiratory quinol oxidase was cloned from thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It contains three genes, soxA, soxB and soxC. The first two genes code for proteins related to the cytochrome c oxidase subunits II and I, respectively. soxC encodes a protein homologous to cytochrome b, which is a subunit of the mitochondrial and bacterial cytochrome c reductases and the chloroplast cytochrome b6f complex. soxA is preceded by a promoter and the genes are cotranscribed into a 4 kb mRNA. Their protein products form a complex which has been partially purified and has quinol oxidase activity. The reduced minus oxidized absorption spectrum of the complex has two maxima at 586 and 606 nm. The latter is typical of cytochrome c oxidase. The complex contains four haems A. Two haems belong to the 'cytochrome oxidase' part of the complex and two are probably bound to be apocytochrome b (SoxC) and responsible for the 586 nm absorption peak. The homology between the sox gene products and their mitochondrial counterparts suggests that energy conservation coupled to the quinol oxidation catalysed either by the Sulfolobus oxidase or two mitochondrial respiratory enzymes may have a similar mechanism.

Amino Acid Sequence↗