FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1990989076> ?p ?o ?g. }

• W1990989076 endingPage "25741" @default.
• W1990989076 startingPage "25735" @default.
• W1990989076 abstract "Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool. Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool. Leigh syndrome (LS) 3The abbreviations used are: LSLeigh syndromeCOXcytochrome c oxidaseHPLChigh performance liquid chromatographyITCisothermal titration calorimetrySUsubunit(s). 3The abbreviations used are: LSLeigh syndromeCOXcytochrome c oxidaseHPLChigh performance liquid chromatographyITCisothermal titration calorimetrySUsubunit(s). is an autosomal recessive inherited neurodegenerative disorder characterized by focal, bilateral lesions in one or more areas of the central nervous system (1Leigh D. J. Neurol. Neurosurg. Psychiatry. 1951; 14: 216-221Crossref PubMed Scopus (633) Google Scholar). Symptoms start in early childhood, and the disease usually progresses rapidly. Although mutations in various mitochondrial enzymes can result in LS, its most frequent trigger is deficiency of cytochrome c oxidase (COX) caused by mutations in the SURF1 gene, as identified in LS patients (2Tiranti V. Hoertnagel K. Carrozzo R. Galimberti C. Munaro M. Granatiero M. Zelante L. Gasparini P. Marzella R. Rocchi M. Bayona-Bafaluy M.P. Enriquez J.A. Uziel G. Bertini E. Dionisi-Vici C. Franco B. Meitinger T. Zeviani M. Am. J. Hum. Genet. 1998; 63: 1609-1621Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 3Zhu Z. Yao J. Johns T. Fu K. De Bie I. Macmillan C. Cuthbert A.P. Newbold R.F. Wang J. Chevrette M. Brown G.K. Brown R.M. Shoubridge E.A. Nat. Genet. 1998; 20: 337-343Crossref PubMed Scopus (538) Google Scholar). Human SURF1, the first gene of the SURFEIT gene locus on chromosome 9, encodes a 30-kDa protein related to COX assembly (2Tiranti V. Hoertnagel K. Carrozzo R. Galimberti C. Munaro M. Granatiero M. Zelante L. Gasparini P. Marzella R. Rocchi M. Bayona-Bafaluy M.P. Enriquez J.A. Uziel G. Bertini E. Dionisi-Vici C. Franco B. Meitinger T. Zeviani M. Am. J. Hum. Genet. 1998; 63: 1609-1621Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 3Zhu Z. Yao J. Johns T. Fu K. De Bie I. Macmillan C. Cuthbert A.P. Newbold R.F. Wang J. Chevrette M. Brown G.K. Brown R.M. Shoubridge E.A. Nat. Genet. 1998; 20: 337-343Crossref PubMed Scopus (538) Google Scholar). Leigh syndrome cytochrome c oxidase high performance liquid chromatography isothermal titration calorimetry subunit(s). Leigh syndrome cytochrome c oxidase high performance liquid chromatography isothermal titration calorimetry subunit(s). Mitochondrial COX consists of up to 13 subunits (SU). The three core SU encoded by the mitochondrial genome carry all of the redox-active cofactors, two heme a moieties, and three copper ions. These three SU are highly conserved among different organisms and represent the main components of bacterial oxidase complexes as well (4Belevich I. Verkhovsky M.I. Antioxid. Redox Signal. 2008; 10: 1-29Crossref PubMed Scopus (117) Google Scholar, 5Pereira M.M. Santana M. Teixeira M. Biochim. Biophys. Acta. 2001; 1505: 185-208Crossref PubMed Scopus (384) Google Scholar). The assembly process of mitochondrial COX is only marginally understood, involving the interplay of a large number of auxiliary proteins (6Fontanesi F. Soto I.C. Horn D. Barrientos A. Am. J. Physiol. Cell Physiol. 2006; 291: C1129-C1147Crossref PubMed Scopus (193) Google Scholar, 7Herrmann J.M. Funes S. Gene. 2005; 354: 43-52Crossref PubMed Scopus (104) Google Scholar, 8Khalimonchuk O. Rödel G. Mitochondrion. 2005; 5: 363-388Crossref PubMed Scopus (110) Google Scholar, 9Zee J.M. Glerum D.M. Biochem. Cell Biol. 2006; 84: 859-869Crossref PubMed Google Scholar). Despite intensive efforts over more than a decade to unravel Surf1 function, its exact role in COX assembly still remains unclear. Surf1 is not strictly essential for COX assembly because patients with LS have residuals of assembled oxidase with remaining activity of approximately 10–20% in all tissues (2Tiranti V. Hoertnagel K. Carrozzo R. Galimberti C. Munaro M. Granatiero M. Zelante L. Gasparini P. Marzella R. Rocchi M. Bayona-Bafaluy M.P. Enriquez J.A. Uziel G. Bertini E. Dionisi-Vici C. Franco B. Meitinger T. Zeviani M. Am. J. Hum. Genet. 1998; 63: 1609-1621Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, 3Zhu Z. Yao J. Johns T. Fu K. De Bie I. Macmillan C. Cuthbert A.P. Newbold R.F. Wang J. Chevrette M. Brown G.K. Brown R.M. Shoubridge E.A. Nat. Genet. 1998; 20: 337-343Crossref PubMed Scopus (538) Google Scholar). Located in the inner mitochondrial membrane, Surf1 is predicted to form two transmembrane helices connected by a long loop facing the intermembrane space (10Mashkevich G. Repetto B. Glerum D.M. Jin C. Tzagoloff A. J. Biol. Chem. 1997; 272: 14356-14364Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 11Yao J. Shoubridge E.A. Hum. Mol. Genet. 1999; 8: 2541-2549Crossref PubMed Scopus (99) Google Scholar). Sequence alignments confirm the presence of Surf1 homologs in many eukaryotes and prokaryotes (12Poyau A. Buchet K. Godinot C. FEBS Lett. 1999; 462: 416-420Crossref PubMed Scopus (38) Google Scholar). One of the best studied Surf1 proteins is the yeast homolog Shy1p, which has been discovered and characterized in the context of pet mutants (10Mashkevich G. Repetto B. Glerum D.M. Jin C. Tzagoloff A. J. Biol. Chem. 1997; 272: 14356-14364Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Deletion of the gene leads to a strongly decreased COX level, although the residual enzyme appears fully functional. This points to a role of Shy1p in assembly or stabilization of COX (13Nijtmans L.G. Artal Sanz M. Bucko M. Farhoud M.H. Feenstra M. Hakkaart G.A. Zeviani M. Grivell L.A. FEBS Lett. 2001; 498: 46-51Crossref PubMed Scopus (59) Google Scholar), most likely during the formation of an early assembly intermediate consisting of the highly conserved core SU I and II (14Barrientos A. Korr D. Tzagoloff A. EMBO J. 2002; 21: 43-52Crossref PubMed Scopus (141) Google Scholar). So far, only three bacterial homologs have been inspected in closer detail (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar, 16Smith D. Gray J. Mitchell L. Antholine W.E. Hosler J.P. J. Biol. Chem. 2005; 280: 17652-17656Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In Paracoccus denitrificans, two Surf1 homologs were identified and named Surf1c and Surf1q for their specific role in serving a heme aa3-type COX and a related heme ba3-type quinol oxidase, respectively (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar). With the function of Surf1 in COX assembly still being speculative, a role in heme a insertion into COX SU I seemed conceivable (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar, 16Smith D. Gray J. Mitchell L. Antholine W.E. Hosler J.P. J. Biol. Chem. 2005; 280: 17652-17656Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Here we show that P. denitrificans Surf1c and Surf1q are able to bind heme a both in vivo and in vitro. This novel finding suggests that Surf1 proteins promote heme a insertion into SU I of either cytochrome c oxidase or quinol oxidase. In addition, Surf1 may modulate heme a synthase activity and provide a heme a cofactor pool in a safe, chelated form for COX SU I biogenesis. The surf1c gene was obtained via PCR using Paracoccus genomic DNA as template with the forward primer (5′-TATAAGCTTCATATGGCCCGG(CATCAC)5ATGCGCCGTTACCTGTTCCC-3′) containing the sequence for an N-terminal His10 tag and an NdeI site and the reverse primer (5′-ATGAGCTCTCTAGACTAGAATTGCCGCTGCCTG-3′) specifying a SacI site. The product was NdeI- and SacI-digested and cloned into the Escherichia coli expression plasmid pET22b (Novagen), resulting in pFA48. The surf1q gene was obtained via PCR using Paracoccus genomic DNA as template with the forward primer (5′-TATAAGCTTCATATGGCCCGG(CATCAC)5GTGACCCTGCGCCGGCTGG-3′) containing the sequence for an N-terminal His10 tag and an NdeI site and the reverse primer (5′-ATGAGCTCTCTAGACCTTTGCGCCCGTCAGTCC-3′) adding a SacI site. The product was NdeI- and SacI-digested and cloned into the E. coli expression plasmid pET22b, generating pFA49. After sequencing, Surf1 expression plasmids pFA48 and pFA49 were transformed into E. coli C41(DE3) (17Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1574) Google Scholar) competent cells, generating expression strains FA48 and FA49. For cloning a heme a maturation plasmid, the pEC86 (18Arslan E. Schulz H. Zufferey R. Künzler P. Thöny-Meyer L. Biochem. Biophys. Res. Commun. 1998; 251: 744-747Crossref PubMed Scopus (337) Google Scholar) derivative pGR50 was obtained via PCR using a forward primer (5′-ATGGAAGCCGGCGGCACCTC-3′) and reverse primer (5′-CTCGAGTATAGGGTACCACACGGTGCCTGACTGCGTT-3′), which introduces an Acc65I and an XhoI restriction site downstream of the tet promoter. The farnesyltransferase gene ctaB was amplified via PCR using Paracoccus genomic DNA as template with the forward primer (5′-TCAAGGTGTACAAAGGAGATACTCATGGCCGATATCAACGCATAT-3′) introducing a BsrGI restriction site and a ribosome-binding site and the reverse primer (5′-TAATAGCTCGAGATATGGGTACCTCACCATCCTCCGACCCAG-3′) adding an Acc65I and an XhoI site. The PCR product was BsrGI- and XhoI-digested and cloned into the Acc65I and XhoI sites of pGR50, resulting in the plasmid pGR51. In the next step, the heme a synthase gene ctaA was amplified via PCR using genomic Paracoccus DNA as template with the forward primer (5′-TCAAGGTGTACAAAGGAGATACTCATGTCGCGCCCGATCGAGAAG-3′) again introducing a BsrGI site and a ribosome-binding site and the reverse primer (5′-TAATAGCTCGAGATATGGGTACCTCATCGGACAGTTCCCCG-3′) adding an Acc65I and an XhoI site. This product was ligated into the Acc65I and XhoI sites of pGR51, generating the final heme a maturation plasmid pGR52. After sequencing, pGR52 was transformed into the Surf1 expression strains FA48 and FA49 (see above), resulting in FA48GR52 and FA49GR52, respectively. To introduce the H193A mutation into Surf1c, the mutant primer (5′-GTCGCGGTCGAGGGAATTCCGAACAACGCCCTGAGCTATGCC-3′) specifying the amino acid exchange and an EcoRI site for screening purposes as well as the expression vector pFA48 (see above) as template were used in a QuikChange mutagenesis reaction (Stratagene). Clones were checked for the mutation via restriction analysis and sequencing and were subsequently transformed into E. coli C41(DE3) cells already containing the plasmid pGR52. For the Surf1q H202A mutation, a forward primer (5′-GCGCTGGGCTATGCGGCGACCTGG-3′) containing the sequence for the mutation that also introduces a BseYI site and a reverse primer (5′-GCTGTTGCGAAAACGGACCACGGTCA-3′) were used to amplify the expression plasmid pFA49 (see above) via PCR. After confirming the mutation, a plasmid was transformed into E. coli C41(DE3) cells that already contained the plasmid pGR52. Ten-liter E. coli cultures were grown on rich medium containing antibiotics at 32 °C in baffled flasks. Protein expression was induced with 1 mm final concentration isopropyl β-d-thiogalactopyranoside at A600 = 1.0, and cells were harvested after 3.5 h. Membranes were prepared by established methods, and protein concentration was determined using a modified Lowry protocol (19Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar, 20Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5319) Google Scholar). Expression of heme a-synthesizing enzymes was checked by acidic acetone/ether extraction (21Weinstein J.D. Beale S.I. J. Biol. Chem. 1983; 258: 6799-6807Abstract Full Text PDF PubMed Google Scholar) of heme from a small portion of the E. coli culture. After evaporation of the ether, the heme preparation was dissolved in 200 μl of dimethyl sulfoxide and analyzed by subsequent pyridine spectra of the extract (see below). A heme a solution was prepared from purified COX (22Hendler R.W. Pardhasaradhi K. Reynafarje B. Ludwig B. Biophys. J. 1991; 60: 415-423Abstract Full Text PDF PubMed Scopus (93) Google Scholar) as mentioned above, and the concentration was determined spectroscopically. Membranes were solubilized in the presence of 3% (w/v) Triton X-100 in buffer (50 mm sodium phosphate, pH 8, 300 mm NaCl) at a final protein concentration of 10 mg/ml. The solubilisate was loaded on a nickel-nitrilotriacetic acid column (Qiagen), and Triton X-100 was exchanged against n-dodecyl-β-d-maltoside by washing with 10 column volumes of buffer (as above) containing 0.02% (w/v) n-dodecyl-β-d-maltoside and 20 mm imidazole. After washing the column with 4 column volumes of buffer containing 50 mm imidazole and 80 mm imidazole each, His-tagged Surf1 was eluted from the column by washing with 5 column volumes of buffer containing 200 mm imidazole. For isothermal titration calorimetry (ITC) measurements (see below), Surf1 proteins were purified further by size-exclusion chromatography on an Äkta purifier system (GE Healthcare) using a Superose 6 column equilibrated with 20 mm sodium phosphate, pH 8, 150 mm NaCl, 0.02% (w/v) n-dodecyl-β-d-maltoside. Redox difference spectra were recorded in the visible range on a Hitachi U-3000 spectrometer. Native and denaturing spectra were recorded by established methods (23Ludwig B. Schatz G. Proc. Natl. Acad. Sci. U.S.A. 1980; 77: 196-200Crossref PubMed Scopus (223) Google Scholar), and heme a concentration was determined using the extinction coefficient ϵ587–620 nm = 21.7 cm−1m−1 for heme a (24Williams Jr., J.N. Arch. Biochem. Biophys. 1964; 107: 537-543Crossref PubMed Scopus (283) Google Scholar). For SDS-PAGE, samples were denatured in SDS-containing buffer for 20 min at 37 °C. Electrophoresis was performed on 12% polyacrylamide gels according to Laemmli (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar). For heme staining, samples were incubated with lithium dodecyl sulfate-PAGE sample buffer on ice. Lithium dodecyl sulfate electrophoresis was performed overnight at 4 °C on 12% polyacrylamide gels (26Delepelaire P. Chua N.H. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 111-115Crossref PubMed Google Scholar), and proteins were blotted on polyvinylidene fluoride membranes. After heme staining (27Dutta C. Henry H.L. Anal. Biochem. 1990; 184: 96-99Crossref PubMed Scopus (17) Google Scholar), membranes were destained with methanol, and proteins were stained with Ponceau S. ITC measurements were carried out on a VP-ITC ultrasensitive titration calorimeter (MicroCal LLC, Northampton, MA). Protein samples were dialyzed twice for several hours at 4 °C against measuring buffer (see Table 1) and centrifuged (20,000 × g, 15 min, 4 °C) to remove insoluble material. ITC titrations and data analyses were performed as described (28Anderka O. Loenze P. Klabunde T. Dreyer M.K. Defossa E. Wendt K.U. Schmoll D. Biochemistry. 2008; 47: 4683-4691Crossref PubMed Scopus (25) Google Scholar).TABLE 1Thermodynamic parameters for heme a binding to Surf1 proteins determined by ITCNaBinding stoichiometry.KdΔHTΔSnMkcal/molkcal/molSurf1c WT0.9 ± 0.0303 ± 28−21.1 ± 0.1−12.3 ± 0.1Surf1q WT0.9 ± 0.1650 ± 40−11.6 ± 0.2−3.2 ± 0.3Surf1c H193A0.8 ± 0.23253 ± 1049−10.5 ± 0.8−2.9 ± 1.0Surf1q H202A0.9 ± 0.091650 ± 543−11.9 ± 2.0−4.0 ± 2.2a Binding stoichiometry. Open table in a new tab In earlier studies of our group, we showed that the two P. denitrificans homologs Surf1c and Surf1q act exclusively on their cognate oxidase (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar). Here, we focus on the heterologous expression and characterization of the two Surf1 homologs to elucidate their exact role in oxidase biogenesis. Expression in E. coli was chosen for the fact that this bacterium neither carries a Surf1 homolog nor provides a heme a-synthesizing machinery and therefore allows a clear-cut analysis of potential interactions between these components. Heme a is synthesized from heme b in two steps. First, heme b is converted into heme o via farnesylation, which is catalyzed by the heme o synthase CtaB (29Mogi T. Saiki K. Anraku Y. Mol. Microbiol. 1994; 14: 391-398Crossref PubMed Scopus (90) Google Scholar). The second step is catalyzed by heme a synthase CtaA not encoded in the E. coli genome. This latter reaction is oxygen-dependent because it involves conversion of a methyl group to a formyl group (29Mogi T. Saiki K. Anraku Y. Mol. Microbiol. 1994; 14: 391-398Crossref PubMed Scopus (90) Google Scholar). Because the endogenous E. coli CtaB homolog CyoE may not interact with the Paracoccus CtaA, both Paracoccus enzymes CtaA and CtaB were expressed heterologously under high aeration (see “Experimental Procedures”), and heme a content of the E. coli cells was verified by pyridine redox spectra. In contrast to E. coli C41(DE3) cells or strains that expressed Surf1c or Surf1q alone, strains containing Paracoccus CtaA and CtaB in trans exhibited the characteristic heme a peak at 587 nm. Thus, the Paracoccus enzymes CtaA and CtaB were expressed in a functional state, enabling these E. coli strains to synthesize heme a. Paracoccus Surf1 proteins were inducibly expressed in E. coli either in the absence or presence of constitutively expressed Paracoccus CtaA and CtaB. Both Surf1 proteins were incorporated into the E. coli cytoplasmic membrane as verified by cell fractionation and Western blot analysis. Membranes were solubilized, and the recombinant Surf1 proteins were purified by affinity chromatography via their N-terminal His10 tag; as shown previously, the tag does not interfere with Surf1 function (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar). The resulting preparations contained no major impurities (Fig. 1A), and both proteins were produced with final yields between 1 and 2 mg of purified protein/liter of medium. After expression in the absence of the heme a biosynthesis machinery, colorless preparations of Surf1 were obtained. However, when expressed in the presence of CtaA and CtaB, Surf1 displayed a greenish-brown color. Heme and protein staining after electrophoresis in semidenaturing gels (Fig. 1B) showed that heme was associated with both Surf1c and Surf1q and was lost after the addition of SDS. Pyridine redox spectra demonstrated that purified Surf1c contained heme a, which was identified by a prominent peak at 587 nm (Fig. 2A). In addition, a minor peak at 552 nm was observed, which corresponds to heme o as verified by HPLC. In native redox spectra (Fig. 2C), the absorption maximum in the α region was at 595 nm for heme a, whereas heme o appeared as a shoulder around 560 nm. The molar heme a/protein ratio was 0.17 ± 0.05 (mean ± S.D. from four independent preparations). Pyridine redox spectra of purified Surf1c confirmed the consistent lack of heme a in protein preparations that were expressed in the absence of CtaA and CtaB (Fig. 2A). Likewise, when expressed in the presence of CtaA and CtaB, Surf1q contained heme a, as indicated by a prominent peak at 587 nm under denaturing conditions (Fig. 2B), in addition to an absorption peak at around 552 nm identified as heme o by HPLC. In native redox spectra (Fig. 2C), the absorption maximum of the detected heme a in the α region was at 600 nm, and the heme o peak appeared as a shoulder at 560 nm. The heme a/protein ratio was 0.09 ± 0.03 (mean ± S.D. from three independent preparations), only about half the value obtained for Surf1c. Spectra of Surf1q preparations expressed in the absence of CtaA and CtaB lacked a heme a signal, and only traces of heme o were detected (Fig. 2B). Sequence alignments of a number of Surf1 homologs from different species show several conserved amino acid residues, most of which are clustered around the periplasmic sides of the two predicted transmembrane helices (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar). To address the question of which residues of the protein are involved in heme a binding, we focused on a highly conserved histidine in the C-terminal helix and exchanged it for an alanine (Surf1c H193A and Surf1q H202A). The two mutant proteins were expressed in the presence of CtaA and CtaB and purified like their wild-type counterparts with similar expression rates and yields. Prior to purification, the cell cultures expressing the Surf1 mutants tested positive for functional heme a synthesis. After purification, heme staining (Fig. 1B) and spectral analysis (Fig. 2) demonstrated a lack of heme a in either variant, and only traces of heme o were detected in the Surf1c mutant protein. The complete absence of heme a indicated that the conserved histidine residue is required for heme a binding of Surf1. A heme a solution was titrated with either apo-Surf1c or apo-Surf1q, i.e. Surf1 proteins that had been expressed in the absence of CtaA and CtaB. The titration led to a distinct spectral red shift of the heme signal, most noticeable in its oxidized form, from 412 to 419 nm (Fig. 3). This shift was assigned to the specific binding of heme a to Surf1c and Surf1q because spectra of both Surf1 expressed in the presence of CtaA and CtaB also exhibited an absorption maximum at 419 nm in their oxidized state. However, this spectral shift did not occur with either of the histidine mutants of Surf1, suggesting that this side chain is indeed required to ligand the heme a metal ion (Fig. 3). The in vitro binding of heme a to apo-Surf1c and apo-Surf1q was quantified using ITC. All titrations yielded a binding stoichiometry near unity (Fig. 4 and Table 1). For heme binding to the wild-type proteins, submicromolar affinities were measured, with Surf1c (Kd = 303 nm) showing a higher affinity compared with Surf1q (Kd = 650 nm). Binding was strongly exothermic with an entropic cost. Remarkably, Surf1c exhibited a negative ΔH value approximately twice as large as observed for Surf1q (−21.1 versus −11.6 kcal/mol). This suggests that the superior heme a-binding affinity of Surf1c may be due to additional polar interactions that are reflected by the strongly increased binding enthalpy (30Holdgate G.A. BioTechniques. 2001; 31 (168–170): 164-166PubMed Google Scholar). Alanine substitution of the conserved histidine residue (see above) affected heme a binding, especially in the case of Surf1c (Fig. 4B). Here, binding affinity was reduced by a factor of ∼11; for Surf1q, affinity was decreased by a factor of ∼3. For Surf1c H193A, a strongly altered binding enthalpy was observed (−10.5 versus −21.1 kcal/mol for the wild type), which was in the same range as ΔH observed for the Surf1q protein. By contrast, the Surf1q H202A variant showed only minor changes in the thermodynamic profile relative to wild-type Surf1q. These observations suggest that the conserved histidine residue plays a more stringent role in heme a binding in the case of Surf1c. The strong change in ΔH for the H193A variant of Surf1c relative to the wild type can be tentatively explained by the loss of polar interactions of the heme ligand with the histidine residue. Defects in the biogenesis pathway of COX are frequently associated with severe respiratory deficiencies. Mitochondrial COX is a multisubunit respiratory chain complex, and correct assembly of SU and redox centers is crucial for its function. More than 30 proteins are involved in the biogenesis of COX in eukaryotes, whereas only five assembly proteins are found in bacteria, all of the latter possibly involved in cofactor delivery (31Greiner P. Hannappel A. Werner C. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 904-911Crossref PubMed Scopus (28) Google Scholar). One of them is Surf1, which has been associated with LS, a fatal neurodegenerative disorder in humans. In this study, we have focused on the characterization of the two homologous Surf1 proteins of Paracoccus, Surf1c and Surf1q, to gain insight into their role in oxidase assembly. To our knowledge, this is the first study to investigate purified Surf1 proteins in detail. Contrary to concepts ranging from stabilizing mature SU I to an involvement in copper homeostasis (32Barrientos A. Gouget K. Horn D. Soto I.C. Fontanesi F. Biochim. Biophys. Acta. 2009; 1793: 97-107Crossref PubMed Scopus (85) Google Scholar, 33Stiburek L. Vesela K. Hansikova H. Hulkova H. Zeman J. Am. J. Physiol. Cell Physiol. 2009; 296: C1218-C1226Crossref PubMed Scopus (73) Google Scholar), both proteins were found to bind heme a at an equimolar ratio and with submicromolar affinities. Heme-binding affinities of a bacterial heme uptake machinery were reported to be in the same range (34Izadi-Pruneyre N. Huché F. Lukat-Rodgers G.S. Lecroisey A. Gilli R. Rodgers K.R. Wandersman C. Delepelaire P. J. Biol. Chem. 2006; 281: 25541-25550Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 35Krieg S. Huché F. Diederichs K. Izadi-Pruneyre N. Lecroisey A. Wandersman C. Delepelaire P. Welte W. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 1045-1050Crossref PubMed Scopus (121) Google Scholar). Interestingly, the affinity of Surf1c for heme a is about twice as large as that of Surf1q (303 versus 650 nm). COX binds two heme a molecules, whereas the quinol oxidase accommodates one heme b and one heme a. Therefore, more heme a is needed for the assembly of COX. As Surf1c and Surf1q specifically supply heme a to their corresponding oxidases (15Bundschuh F.A. Hoffmeier K. Ludwig B. Biochim. Biophys. Acta. 2008; 1777: 1336-1343Crossref PubMed Scopus (29) Google Scholar), we envisage this difference in heme a affinity as a potential regulatory mechanism to ensure proper heme a distribution between the two oxidases. The two Surf1 proteins seem to provide slightly different binding sites for heme a, which is reflected both by the different absorption maxima in the α region of redox spectra (595 nm for Surf1c versus 600 nm for Surf1q; see Fig. 2C) and by the difference in ΔH values for the heme binding (−21.1 kcal/mol for Surf1c versus −11.6 kcal/mol for Surf1q). In addition, the substitution of the conserved histidine has a much stronger effect on heme binding for Surf1c than for Surf1q. We used E. coli as a heterologous host and introduced the heme a biosynthesis machinery derived from Paracoccus CtaA (heme a synthase; COX15 in eukaryotes) and CtaB (heme o synthase; COX10 in eukaryotes). The in vivo level of heme binding of heterologously expressed Surf1 proteins in the presence of CtaA and CtaB changed with growth conditions. Aeration had an especially strong influence on heme content, which is easily explained by the oxygen dependence of CtaA (see above). Therefore, heme content varied with different preparations, although never reaching a 1:1 heme/protein ratio. In our experimental system, CtaA and CtaB were expressed constitutively in contrast to the inducible overexpression of the Surf1 proteins, and it may be assumed that heme a synthase activity could not keep up with Surf1 expression rate. In this context, the observed heme o binding to the Surf1 proteins is probably unspecific and caused primarily by the abundance of heme o in the E. coli host, where heme o is required for the bo3-type terminal oxidase. Preliminary experiments with both Surf1 proteins isolated from Paracoccus show that they exclusively bind heme a as ligand in the native host. This is possibly due to the low levels of the heme o intermediate in Paracoccus (36de Gier J.W. Lübben M. Reijnders W.N. Tipker C.A. Slotboom D.J. van Spanning R.J. Stouthamer A.H. van der Oost J. Mol. Microbiol. 1994; 13: 183-196Crossref PubMed Scopus (110) Google Scholar). It is generally agreed that heme a does not occur in a protein-free form in the membrane because it is detrimental to the cell (37Mense S.M. Zhang L. Cell Res. 2006; 16: 681-692Crossref PubMed Scopus (209) Google Scholar). Therefore, in eukaryotes, a direct heme a transfer from COX15 to COX SU I was assumed. COX15 activity and expression were found to be highly regulated in yeast, especially by heme b, which is not only a precursor of heme a but also the cofactor of heme a synthase (38Wang Z. Wang Y. Hegg E.L. J. Biol. Chem. 2009; 284: 839-847Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In the same study, it was proposed that heme a liberation from COX15 could be regulated by SU I or by COX assembly intermediates because this would prevent uncontrolled release of potentially toxic heme a. Our present data strongly suggest that Surf1 interacts with heme a synthase and may provide a heme a pool function, which directs heme a flux from heme a synthase to its final target SU I. As proposed in Fig. 5, membrane-integral Surf1 interacts with heme a synthase to take over the heme a product from its active site and then dissociates from the enzyme to allow continuation of heme a synthesis. Surf1 is suggested to interact directly with COX SU I to eventually transfer both heme groups present in the mature form of the enzyme. At the moment, we can only speculate on the mechanism of heme a insertion into SU I. Studies on heme incorporation into four-helix bundles mimicking oxidase structure resulted in heme a-binding affinities in the low nanomolar Kd range (39Gibney B.R. Isogai Y. Rabanal F. Reddy K.S. Grosset A.M. Moser C.C. Dutton P.L. Biochemistry. 2000; 39: 11041-11049Crossref PubMed Scopus (73) Google Scholar); this high affinity would readily allow transfer of heme a because Surf1 binds heme a only with high nanomolar Kd values. We propose that heme a is inserted into SU I in a co-translational manner (Fig. 5). The 12-transmembrane helix bundle structure of SU I appears as a fairly rigid scaffold that, once folded, may not allow access to the rather bulky heme a moiety, as much as the fully assembled oxidase complex hardly loses either of its heme groups, not even on prolonged and excessive purification procedures. The yeast homolog of Surf1, Shy1p, has been found to interact with Mss51p and Cox14p, two mitochondrial COX assembly factors involved in the regulation of SU I expression (40Barrientos A. Zambrano A. Tzagoloff A. EMBO J. 2004; 23: 3472-3482Crossref PubMed Scopus (165) Google Scholar, 41Perez-Martinez X. Broadley S.A. Fox T.D. EMBO J. 2003; 22: 5951-5961Crossref PubMed Scopus (153) Google Scholar). For the yeast system, it was suggested that Shy1p acts downstream of Mss51p and Cox14p, taking over and stabilizing fully assembled SU I, and thus allows its association with additional SU and assembly factors (42Pierrel F. Bestwick M.L. Cobine P.A. Khalimonchuk O. Cricco J.A. Winge D.R. EMBO J. 2007; 26: 4335-4346Crossref PubMed Scopus (116) Google Scholar) and later the bc1 complex to form supercomplexes (43Mick D.U. Wagner K. van der Laan M. Frazier A.E. Perschil I. Pawlas M. Meyer H.E. Warscheid B. Rehling P. EMBO J. 2007; 26: 4347-4358Crossref PubMed Scopus (105) Google Scholar). However, Mss51p and Cox14p are only found in fungi and lack counterparts both in higher eukaryotes and in bacteria; moreover, compared with other Surf1 homologs, Shy1p has a large additional loop not conserved in Surf1 proteins of other species. Because heme a incorporation into SU I is likely to occur co-translationally, an interaction of Shy1p with the translation machinery for SU I in yeast does not contradict its proposed role in heme incorporation. The observed association with SU I in later stages of the assembly process (13Nijtmans L.G. Artal Sanz M. Bucko M. Farhoud M.H. Feenstra M. Hakkaart G.A. Zeviani M. Grivell L.A. FEBS Lett. 2001; 498: 46-51Crossref PubMed Scopus (59) Google Scholar, 42Pierrel F. Bestwick M.L. Cobine P.A. Khalimonchuk O. Cricco J.A. Winge D.R. EMBO J. 2007; 26: 4335-4346Crossref PubMed Scopus (116) Google Scholar, 43Mick D.U. Wagner K. van der Laan M. Frazier A.E. Perschil I. Pawlas M. Meyer H.E. Warscheid B. Rehling P. EMBO J. 2007; 26: 4347-4358Crossref PubMed Scopus (105) Google Scholar) hints at an additional role of Shy1p. To summarize the possible functions of Surf1 in COX biogenesis, a 3-fold contribution to heme a insertion seems plausible. Surf1 (i) may modulate heme synthase activity by abstracting the product heme a from the active site of CtaA and thus allows continuing heme synthesis; (ii) avoids the presence and presumed detrimental action of free heme a in the membrane by providing a safe, yet readily available pool of this cofactor; and (iii) specifically chaperones heme a to its target sites in SU I of COX and positions the bulky cofactor for presumed co-translational insertion into both sites within the 12-transmembrane helix scaffold of COX SU I. The importance of Surf1 in COX biogenesis is exemplified by the fact that its absence leads to severe COX deficiency in humans, which causes neurodegenerative LS. The elucidation of the exact function of Surf1 may spur the development of a treatment for LS. Our findings both in vivo and in vitro that Surf1 is a heme a-binding protein strongly indicate that Surf1 is more likely involved in direct cofactor incorporation into COX SU I rather than merely being a regulatory protein. We thank Dieter Schmoll (Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany) for granting access to the ITC instrument, Oliver-Matthias H. Richter for helpful discussions and critical comments on the manuscript, Peter Greiner for planning the cloning of pGR52, and Hans-Werner Müller and Andrea Herrmann for excellent technical assistance." @default.
• W1990989076 created "2016-06-24" @default.
• W1990989076 creator A5016095609 @default.
• W1990989076 creator A5022707818 @default.
• W1990989076 creator A5031513588 @default.
• W1990989076 creator A5082310204 @default.
• W1990989076 date "2009-09-01" @default.
• W1990989076 modified "2023-10-03" @default.
• W1990989076 title "Surf1, Associated with Leigh Syndrome in Humans, Is a Heme-binding Protein in Bacterial Oxidase Biogenesis" @default.
• W1990989076 cites W1481324524 @default.
• W1990989076 cites W1515235319 @default.
• W1990989076 cites W1576988856 @default.
• W1990989076 cites W1775749144 @default.
• W1990989076 cites W1966683225 @default.
• W1990989076 cites W1968048466 @default.
• W1990989076 cites W1974032935 @default.
• W1990989076 cites W1974260422 @default.
• W1990989076 cites W1989584376 @default.
• W1990989076 cites W1998295783 @default.
• W1990989076 cites W2001940147 @default.
• W1990989076 cites W2006159932 @default.
• W1990989076 cites W2012005944 @default.
• W1990989076 cites W2012560500 @default.
• W1990989076 cites W2015387867 @default.
• W1990989076 cites W2018204791 @default.
• W1990989076 cites W2023406366 @default.
• W1990989076 cites W2031000509 @default.
• W1990989076 cites W2031423487 @default.
• W1990989076 cites W2033671245 @default.
• W1990989076 cites W2034601563 @default.
• W1990989076 cites W2038382454 @default.
• W1990989076 cites W2040997463 @default.
• W1990989076 cites W2041315905 @default.
• W1990989076 cites W2046180227 @default.
• W1990989076 cites W2059787437 @default.
• W1990989076 cites W2060703619 @default.
• W1990989076 cites W2067218778 @default.
• W1990989076 cites W2091263922 @default.
• W1990989076 cites W2091381823 @default.
• W1990989076 cites W2091807016 @default.
• W1990989076 cites W2093785402 @default.
• W1990989076 cites W2098475709 @default.
• W1990989076 cites W2100837269 @default.
• W1990989076 cites W2101871915 @default.
• W1990989076 cites W2120587654 @default.
• W1990989076 cites W2123634642 @default.
• W1990989076 cites W2124287644 @default.
• W1990989076 cites W2135136314 @default.
• W1990989076 cites W2149901817 @default.
• W1990989076 cites W2150245289 @default.
• W1990989076 cites W2166258960 @default.
• W1990989076 doi "https://doi.org/10.1074/jbc.m109.040295" @default.
• W1990989076 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2757975" @default.
• W1990989076 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19625251" @default.
• W1990989076 hasPublicationYear "2009" @default.
• W1990989076 type Work @default.
• W1990989076 sameAs 1990989076 @default.
• W1990989076 citedByCount "54" @default.
• W1990989076 countsByYear W19909890762012 @default.
• W1990989076 countsByYear W19909890762013 @default.
• W1990989076 countsByYear W19909890762014 @default.
• W1990989076 countsByYear W19909890762015 @default.
• W1990989076 countsByYear W19909890762016 @default.
• W1990989076 countsByYear W19909890762017 @default.
• W1990989076 countsByYear W19909890762018 @default.
• W1990989076 countsByYear W19909890762020 @default.
• W1990989076 countsByYear W19909890762021 @default.
• W1990989076 countsByYear W19909890762023 @default.
• W1990989076 crossrefType "journal-article" @default.
• W1990989076 hasAuthorship W1990989076A5016095609 @default.
• W1990989076 hasAuthorship W1990989076A5022707818 @default.
• W1990989076 hasAuthorship W1990989076A5031513588 @default.
• W1990989076 hasAuthorship W1990989076A5082310204 @default.
• W1990989076 hasBestOaLocation W19909890761 @default.
• W1990989076 hasConcept C104317684 @default.
• W1990989076 hasConcept C123249941 @default.
• W1990989076 hasConcept C131934819 @default.
• W1990989076 hasConcept C181199279 @default.
• W1990989076 hasConcept C185592680 @default.
• W1990989076 hasConcept C2776217839 @default.
• W1990989076 hasConcept C2778461778 @default.
• W1990989076 hasConcept C28859421 @default.
• W1990989076 hasConcept C501734568 @default.
• W1990989076 hasConcept C55493867 @default.
• W1990989076 hasConcept C86803240 @default.
• W1990989076 hasConceptScore W1990989076C104317684 @default.
• W1990989076 hasConceptScore W1990989076C123249941 @default.
• W1990989076 hasConceptScore W1990989076C131934819 @default.
• W1990989076 hasConceptScore W1990989076C181199279 @default.
• W1990989076 hasConceptScore W1990989076C185592680 @default.
• W1990989076 hasConceptScore W1990989076C2776217839 @default.
• W1990989076 hasConceptScore W1990989076C2778461778 @default.
• W1990989076 hasConceptScore W1990989076C28859421 @default.
• W1990989076 hasConceptScore W1990989076C501734568 @default.
• W1990989076 hasConceptScore W1990989076C55493867 @default.
• W1990989076 hasConceptScore W1990989076C86803240 @default.
• W1990989076 hasIssue "38" @default.
• W1990989076 hasLocation W19909890761 @default.

• next