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Bits and Bytes and Eukaryotes

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Bits and Bytes and Eukaryotes J Mol Med (2000) 78:239–242 © Springer-Verlag 2000 Digital Object Identifier (DOI) 10.1007/s001099900110 Yurii V. Chinenov Cytochrome c oxidase assembly factors with a Bioinformatics: thioredoxin fold are conserved among prokaryotes Bits and Bytes and eukaryotes Abstract Cytochrome c oxidase transport function. Reported sequence (COX) is a multi-subunit terminal oxi- similarities, together with the function- dase of the eukaryotic respiratory chain al role of bacterial Sco-related proteins involved in the reduction of oxygen to suggest that Sco-related proteins re- water. Numerous lines of evidence sug- present a new class of membrane- gesting that assembly of mitochondrial gest that the assembly of COX is a anchored thiol:disulfide oxidoreduc- complexes is dependent on a number multi-step, assisted process that de- tases involved in COX maturation. of accessory proteins [5, 6]. Assembly pends on several assembly factors with factors may be responsible for the largely unknown functions. Sco1/2 Key words Sco · Cytochrome transport and insertion of heme proteins have been isolated as high- c oxidase · Peroxiredoxins · (Cox10p, Cox11p), copper (Cox17p), copy number suppressors of a deletion Thioredoxin fold · Cox17 or magnesium (Cox11p) into inactive of copper chaperone Cox17, implicat- enzymes, intermembrane subunit trans- ing Sco1/2 in copper transport to COX Abbreviations COX: Cytochrome port, and folding of COX subunits subunits I or II. Here I report the simi- c oxidase [7, 8, 9, 10, 11, 12]. larity of Sco1/2 assembly factors to pe- Sco1p and the related Sco2p were roxiredoxins and thiol:disulfide oxido- originally identified as proteins re- reductases with a thioredoxin fold, sug- quired for COX assembly. Also, they gesting that Sco-related proteins per- Cytochrome c oxidase (COX) is the are capable of partial suppressing form a catalytic rather than a copper terminal oxidase of the respiratory COX17 deletion at elevated concentra- chain catalyzing the reduction of mo- tions of copper [7, 8, 9, 10, 11]. lecular oxygen to water with concomi- Cox17p is an 8-kDa protein containing Y.V. Chinenov (✉) tant oxidation of cytochrome c. COX is a highly labile binuclear Cu (I)-thiolate Howard Hughes Medical Institute, a multi-subunit complex localized in cluster, presumably involved in copper University of Michigan Medical Center, the inner membrane of mitochondria or delivery to COX II subunit [4, 7]. De- 4570 MSRB II, aerobic bacteria. Subunits I–III are letion of SCO1, but not SCO2, in yeast 1150 W. Medical Center Drive, conserved between prokaryotes and results in rapid degradation of both cat- Ann Arbor, MI 48109-0650, USA eukaryotes, and at least two of them alytic subunits of COX. The presence e-mail: [email protected] (I and II) are required for catalytic ac- of CXXXC motif, similar to the CoxII Tel.: +1-734-6152576 tivity [1]. Additional eukaryotic sub- copper binding site, led to the hypothe- Fax: +1-734-6153397 units may be involved in the modula- sis that Sco1p is also directly involved tion of activity, assembly, or tissue spe- in copper transport [10, 12]. Substitu- cific regulation of catalytic functions. tion of either of these cysteine residues Please send articles to: Despite the considerable progress in results in the loss of active COX in Peer Bork elucidating the structural organization yeast [10], demonstrating the impor- Max-Dehlbrück-Center of COX [1] little is known about mech- tance of these residues. However, the for Molecular Medicine (MDC) anisms of assembly of this large enzy- requirement for elevated concentra- Robert-Rössle-Strasse 10 matic complex. The presence of free tions of copper suggests possible alter- D-13122 Berlin, Germany subunits and partially assembled com- native functions of Sco1 and Sco2 in and: plexes suggests that COX assembly is the assembly of COX. EMBL a slow, sequential, and possibly assist- PSI-BLAST [13] searches Meyerhofstrasse 1 ed process [2, 3, 4]. More than 30 com- (E<0.001) against a nonredundant pro- D-69117 Heidelberg, Germany plementation groups deficient in COX tein database revealed a significant E-mail: [email protected] activity have been identified in yeast. similarity of Sco1p and Sco2p to sever- http://www.embl-heidelberg.de/~bork/ Most of these do not associate with al peroxiredoxines with known three- known COX enzymatic activities, sug- dimensional structures and bacterial 240 Fig. 1 Multiple alignment of Sco1-related proteins. A PSI BLAST search (E-value=0.001) yeast Sco1p using PHD and PSIpred of NCBI nonredundant protein database with the C-terminal portion of Saccharomyces algorithms [15, 16] are very similar to cerevisae Sco1p revealed similarity to peroxiredoxins after the second iteration and bacte- that of peroxiredoxins (hORF6 and rat rial thiol:disulfide oxidoreductases after the third iteration. Representative members of HBP23 in Fig. 1) determined by X-ray these groups were aligned using MULTALIN [14] with the following settings: symbol crystallography [17, 18]. The con- comparison table – BLOSUM 62, gap penalty 8, gap penalty at extension 0.05. Aligned se- β quences were prepared for publication using GenDoc [31]. Conserved amino acids were served regions include -strand 3, colored according to the following scheme: dark blue hydrophobic residues α-helix 2, and a loop between them (ACFGHIKLMVWY); light blue aliphatic residues (ILV); gray aromatic residues encompassing active center, β-strand 4, (FHWY); red positively charged residues (KRH); purple DENQ; green polar and hydrophobic residues of β-strand 6. (CDEHKNQRST); yellow small (ACDGNPSTV). Left column SwissProt protein names or Peroxiredoxins and thiol:disulfide GenBank identifier codes; right column amino acid positions are indicated for each pro- oxidoreductases reduce a wide range of tein; on the top of the alignment amino acid positions of aligned proteins; above the align- ment with open rectangles secondary structure predicted with PSIpred [16], α-helixes; substrates, including hydrogen perox- open arrows β-strands. Secondary structure of human AOP2 (ORF6) and rat HBP23 is ide, various organic peroxides, and di- assigned from respective crystal structures and is shown as following: black rectangles sulfide bonds in proteins and low mo- α-helices; black arrows β-strands. The annotation of secondary structure elements is as in lecular weight compounds. The mecha- crystal structure of ORF6 [17, 18]. Orange circles residues of the putative active center nism of substrate reduction involves an and Arg-119 of the ORF6 active center; black stars above the alignment amino acid resi- initial ionization of a cysteine residue dues in the proposed Sco active center that are mutated in patients with infantile cardioen- in the active center (Fig. 1, Cys-25), cephalomyopathy [23] followed by a nucleophilic attack of a substrate and formation of either mixed thiol:disulfide oxidoreductases (Fig. 1). cysteine residues and hydrophobic resi- disulfide bonds or sulfenic acid [17, Although overall similarity is low, sev- dues in the active center (Fig. 1, resi- 19, 20]. The reduction of a mixed di- eral regions in Sco1-related proteins dues 10–30). Representative proteins sulfide bond intermediate involves an and peroxiredoxin/thiol:disulfide oxi- were aligned with MULTALIN [14]. attack by the second cysteine residue in doreductase are conserved, including The secondary structure predictions of the active center leading to the release 241 of a reduced substrate and formation of [24, 25, 26]. Since cytochrome c bio- 5. McEwen JE, Ko C, Kloeckner- intramolecular (thiol:disulfide oxidore- genesis is not affected in sco1 or sco2 Gruissem B, Poyton RO (1986) Nuclear ductase) or intermolecular (2-Cys pero- mutants, the primary target of Sco must functions required for cytochrome c ox- xiredoxins) disulfide bond. Regenera- be elsewhere. The ability of Sco1 to idase biogenesis in Saccharomyces cer- evisiae. Characterization of mutants in tion of the catalytically active reduced suppress cox17 deficiency of copper 34 complementation groups. J Biol form of an enzyme probably involves a transport suggests a possible function Chem 261:11872–11879 thioredoxin/thyoredoxin reductase downstream in the COX assembly 6. Tzagoloff A, Dieckmann CL (1990) system. In peroxiredoxins conserved pathway, probably on the step concom- PET genes of Saccharomyces cerevisi- Arg-119 (Fig. 1) abstracts a proton itant or immediately preceding copper ae. Microbiol Rev 54:211–225 from the catalytic sulfhydryl group insertion. Physical interactions of 7. Srinivasan C, Posewitz MC, George [17]. In hORF6 His-17 stabilizes the Cox17p and Sco1/2p were recently GN, Winge DR (1998) Characterization of the copper chaperone Cox17 of Sac- ionized sulfhydryl group. This position demonstrated in a high throughoutput charomyces cerevisiae. Biochemistry is occupied by an aromatic residue in two-hybrid screening for interacting 37:7572–7577 most analyzed peroxiredoxins and Sco- yeast proteins [27]. Although spontane- 8. Schulze M, Rodel G (1989) Accumula- related proteins, suggesting an alterna- ous incorporation of copper into CoxII tion of the cytochrome c oxidase sub- tive mechanism of thiolate intermedi- CuA center is possible, it requires high units I and II in yeast requires a mito- ate stabilization [18]. In HBP23 inter- concentrations of copper and rather chondrial membrane-associated protein, actions between Arg-119, Glu-28, and nonphysiological pH [28, 29, 30]. The encoded by the nuclear SCO1 gene. Asp-137 coordinating a putative Cl an-
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