Mathias Lübben

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.

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.

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 sub unit 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 (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa3‐type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem‐containing oxidase, called cytochrome bb3, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::KmR) an alternative cyto‐chrome 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.

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 tack 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 it functions in the oxygeNatlon/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.

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 hydro‐dynamically 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 compiex 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 Aa has a 2‐hydroxyethyl geranylgeranyl in position 2 of the porphyrin ring whereas haem A has the related farnesyl‐containing side‐chain.

The respiratory system of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium

The respiratory properties, cellular ATP content and absorption difference spectra of Sulfolobus acidocaldarius (DSM 639) have been investigated. In contrast to earlier postulates regarding Thermoplasma acidophilum [(1984) Syst. Appl. Microbiol. 5, 30‐40], S. acidocaldarius seemed to depend energetically on respiration‐coupled phosphorylation. Its ATP content strictly depended on respiratory activity. Its membrane is capable of proton pumping and presumably contains a branched electron transport system. The latter is composed of at least 2 types of cytochromes, an a+‐ and presumably an o‐type, while c‐type cytochromes could not be detected. It appears possible that one of the terminal oxidases is directly reduced by the socalled caldariellaquinone, found in the same organism [(1983) Syst. Appl. Microbiol. 4, 295‐304].

Redox FTIR difference spectroscopy using caged electrons reveals contributions of carboxyl groups to the catalytic mechanism of haemcopper oxidases

Redox spectra of the haem‐copper oxidases cytochrome aa 3 of Rhodobacter sphaeroides and cytochrome bo 3 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‐finked 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 oxidases.

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 Desulfovibrio 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.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking.

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

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.