The Characterization and Role of Zinc Binding in Yeast Cox4

Yeast Cox4 is a zinc binding subunit of cytochrome c oxidase. Cox4 is the only cofactor-containing subunit that is not directly part of the catalytic core of the enzyme located in the mitochondrial inner membrane. The Zn(II) site is shown to be distinct from the bovine ortholog, as it results from the x-ray structure of the entire cytochrome c oxidase in having a single histidyl residue and three conserved cysteines residues in the coordination sphere. Substitutions at the Cys ligand positions result in non-functional Cox4 proteins that fail to lead to cytochrome oxidase assembly. Limited function exists in His-119 mutants when overexpressed. Zn(II) binding in Cox4 is, therefore, important for the stability of the complex. The solution structure of yeast Cox4 elucidated by multidimensional NMR reveals a C-terminal globular domain consisting of two β sheets analogous to the bovine ortholog except the loop containing the coordinating His in the yeast protein and the fourth Cys in the bovine protein are in different positions in the two structures. The conformation of this loop is dictated by the different sequence position of the fourth coordinating zinc ligand. The Zn(II) ion is buried within the domain, consistent with its role in structural stability. Potential functions of this matrix-facing subunit are discussed. Yeast Cox4 is a zinc binding subunit of cytochrome c oxidase. Cox4 is the only cofactor-containing subunit that is not directly part of the catalytic core of the enzyme located in the mitochondrial inner membrane. The Zn(II) site is shown to be distinct from the bovine ortholog, as it results from the x-ray structure of the entire cytochrome c oxidase in having a single histidyl residue and three conserved cysteines residues in the coordination sphere. Substitutions at the Cys ligand positions result in non-functional Cox4 proteins that fail to lead to cytochrome oxidase assembly. Limited function exists in His-119 mutants when overexpressed. Zn(II) binding in Cox4 is, therefore, important for the stability of the complex. The solution structure of yeast Cox4 elucidated by multidimensional NMR reveals a C-terminal globular domain consisting of two β sheets analogous to the bovine ortholog except the loop containing the coordinating His in the yeast protein and the fourth Cys in the bovine protein are in different positions in the two structures. The conformation of this loop is dictated by the different sequence position of the fourth coordinating zinc ligand. The Zn(II) ion is buried within the domain, consistent with its role in structural stability. Potential functions of this matrix-facing subunit are discussed. Cytochrome c oxidase (CcO), 2The abbreviations used are: CcO, cytochrome c oxidase; IM, inner membrane; WT, wild type; EXAFS, x-ray absorption fine structure; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum correlation; ROS, reactive oxygen species. or complex IV, is the terminal enzyme in the respiratory chain. Complex IV is located in the mitochondria of eukaryotes, where it is embedded within the inner membrane (IM). CcO catalyzes the reduction of molecular oxygen to water (1Yoshikawa S. Adv. Protein Chem. 2002; 60: 341-395Crossref PubMed Scopus (34) Google Scholar). The catalytic cycle of CcO pumps eight protons from the matrix to both the reaction center and into the inner membrane space. Four of the protons are directed to the heme a3-CuB reaction center to participate in the formation of water. The remaining protons are released into the inner membrane space to create a proton gradient, which is used to drive the synthesis of ATP via ATP synthase. The bulk of ATP in eukaryotic cells is produced through the respiratory chain. The importance of the pathway has generated interest in understanding the mechanism of CcO assembly and the function of individual subunits in forming the oligomeric holoenzyme. The subunits of CcO are encoded by both nuclear and mitochondrial genes. The core subunits, Cox1–3, are mitochondrial gene products and either bind cofactors involved in the electron transport pathway or are involved in proton translocation (2Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Hakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1295) Google Scholar, 3Hosler J. Ferguson-Miller S. Mills D.A. Annu. Rev. Biochem. 2006; 75: 165-187Crossref PubMed Scopus (250) Google Scholar). Cox1 contains the heme a, heme a3, and the mononuclear CuB site. One histidine ligand bridges CuB and heme a3, having a role in water formation (4Yoshikawa S. Shinzawa-Itoh K. Tsukihara T. J. Inorg. Biochem. 2000; 82: 1-7Crossref PubMed Scopus (66) Google Scholar). Cox2 contains the binuclear CuA site responsible for accepting the reducing electrons from cytochrome c. Cox3 is a core subunit containing no cofactors and has been proposed to regulate proton uptake through the D-channel (5Gilderson G. Salomonsson L. Aagaard A. Gray J. Brezezinski P. Hosler J. Biochemistry. 2003; 42: 7400-7409Crossref PubMed Scopus (44) Google Scholar). The core CcO complex is surrounded by 10 small peripheral subunits, many of which consist of a single transmembrane helix. The peripheral subunits are encoded by the nuclear genome (6Poyton R.O. McEwen J.E. Annu. Rev. Biochem. 1996; 65: 563-607Crossref PubMed Scopus (434) Google Scholar), but only a subset of these subunits shows sequence similarity between animals and yeast orthologs. Many of these subunits are functionally important, since deletion of genes in yeast for some peripheral subunits results in respiratory deficiency (6Poyton R.O. McEwen J.E. Annu. Rev. Biochem. 1996; 65: 563-607Crossref PubMed Scopus (434) Google Scholar). One conserved peripheral subunit is CoxVb, designated Cox4 in Saccharomyces cerevisiae. Cox4 is an essential subunit of CcO as the enzyme fails to assemble in cox4Δ cells, and core subunits Cox1 and Cox2 fail to accumulate, thus inducing respiratory deficiency (7McEwen J.E. Ko C. Kloeckner-Gruiissem B. Poyton R.O. J. Biol. Chem. 1986; 261: 11872-11879Abstract Full Text PDF PubMed Google Scholar, 12Glerum D.M. Tzagoloff A. FEBS Lett. 1997; 412: 410-414Crossref PubMed Scopus (38) Google Scholar). The importance of CoxVb is apparent from the crystal structure of bovine CcO, as the subunit is closely packed onto Cox1 and Cox3 on the matrix side of the IM (2Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Hakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1295) Google Scholar). CoxVb does not penetrate the lipid bilayer. CoxVb coordinates a single Zn(II) ion through four cysteinyl residues (2Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Hakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1295) Google Scholar). Because the nomenclature for this subunit varies between mammals and yeast, we will use the yeast Cox4 designation for this study as the focus of the work is on the yeast protein. The physiological roles of Cox4 and Zn(II) coordination in CcO remain unclear. Zn(II) has been suggested to be a structural component of the subunit, but this has not been tested. Yeast Cox4 has only three of the four conserved cysteinyl residues in its primary sequence (8Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (523) Google Scholar). The spectroscopic inertness of Zn(II) makes direct determination of its coordination environment difficult, but metal substitution is a useful alternative. Zn(II) prefers tetrahedral coordination geometry. Cd(II) and Co(II) can be substituted in zinc proteins, since they can be accommodated by the same coordination geometry and utilize similar donor ligands as Zn(II). 113Cd(II) has a nuclear spin of ½ and is amenable to NMR analysis. The chemical shift observed of a cadmium nucleus is indicative of the coordination environment. A single one-dimensional measurement can provide clues as to which atoms are directly coordinated to the cadmium nucleus (9Coleman J.E. Methods Enzymol. 1993; 227: 16-43Crossref PubMed Scopus (75) Google Scholar). Co(II) is a useful probe using electronic spectroscopy. Ligand to metal charge transfer bands and absorption in the visible region due to d-d transitions provide information on the coordination environment for both geometry and ligand atoms (10Bertini I. Luchinat C. Eichhorn G.L. Marzilli L.G. Advances in Inorganic Biochemistry. Elsevier Science Publishing Co., Inc., New York1984Google Scholar, 11Maret W. Vallee B.L. Methods Enzymol. 1993; 226: 52-71Crossref PubMed Scopus (206) Google Scholar). We show in this report that yeast Cox4 binds Zn(II) through three cysteinyl residues and a single histidine. The structure of the C-terminal globular domain Cox4 reveals an overall fold similar to that of bovine CoxVb but with local structural difference on the loop accommodating the His ligand. A structural comparison of the conserved hydrophobic residues between the yeast and bovine homologues identify which of them can play a key role in the Cox4-Cox1 interaction. Strains and Vectors—The Δcox4 yeast strain was generously provided by Dr. A. Tzagoloff (12Glerum D.M. Tzagoloff A. FEBS Lett. 1997; 412: 410-414Crossref PubMed Scopus (38) Google Scholar). The wild-type COX4 was amplified from yeast genomic DNA by PCR such that 1000 base pairs upstream of the ATG translation start were included. The primers for the 5′ and 3′ ends of COX4 were 5′-AAAAAGCTTCAGTGTGTATACCCTCC and 5′-AAAGGATCCTATAGATAGAGCAAAGCGTTCG, respectively. The PCR product was then restriction enzyme-digested with HindIII and BamHI for insertion into pRS425 (YEp plasmid with LEU2 selection) and pRS415 (YCp plasmid with LEU2 selection). Mutant COX4 plasmids were constructed by PCR using the WT plasmid as a template. Primers were designed according to the protocol suggested by Stratagene, and the codons selected were based on the optimal frequency in S. cerevisiae. COX4 used for recombinant studies in Escherichia coli was a construct encoding a truncated protein lacking the first 78 residues from the N terminus that form the mitochondrial targeting sequence and an extended segment in the bovine CoxVb structure. The truncated gene was inserted into pET20b+. Mutant variants were generated using the Stratagene QuikChange protocol. The codons selected for mutagenesis were based on the optimal frequency in E. coli. The recombinant had a three residue (MMA) N-terminal extension arising from the PCR protocol. The C-terminal truncate used in the 113Cd NMR and cobalt and zinc titration studies terminated at Asn-75. Yeast Analyses—Transformation of Δcox4 cells was by the lithium acetate procedure. The transformed yeast was grown to stationary phase in the synthetic glucose media to provide strain (+Ura) and plasmid (+Leu) selection. The cells were harvested, spun at 3000 rpm, and subsequently washed with water. mitochondria of transformed yeast were according to the protocol K. G. R. Methods Biol. 65: PubMed Google Scholar). was on with of mitochondrial protein Protein was by the Biochem. PubMed Scopus Google Scholar). The protein was then to The primary was yeast Cox4 and the was The was using and were used as for the and the using Cox4 plasmids were transformed into cells for recombinant protein were grown to an of at using for at The cells were and washed in at The cells were by in containing 10 The was for at with on in between the The was by and through a membrane. The solution was 3000 and with water to the of Cox4 was with an protein using a The was and was with a to The position of Cox4 was by Cox4 were and onto a in at Cox4 as a single with as by analysis. containing Cox4 were and 3000 for The solution of Cox4 was for protein at using an of The zinc was by absorption using a proteins were produced and using the same except the recombinant protein was in The as the and as the The were with at a of The The was for at and absorption were at the on using a of The and x-ray were using and x-ray absorption was as the zinc using an of Methods A. Scopus Google Scholar). were at a of 10 using an and four were The x-ray was by measurement of a zinc metal a of the metal to be The of the extended x-ray absorption fine structure was to be were by using the of phase and functions were using the Rev. Lett. PubMed Scopus Google Scholar). or were on the and was by of Cox4 to a of The solution was on in to the protein from Zn(II). in was and with to The of the was to using The was in a The Cox4 was as the Co(II) complex was was with and to The protein was in a NMR for on a with a The were The frequency used was were with a of and a of a of NMR and were on and at proton of and respectively. the used were with the The and were with a The NMR were on and at and at K. The NMR used for the and the and for structural are in The and of are in The of proton and atoms are and respectively. the and were using the and on through the R. The Scholar). were with the using as the the chemical shift and three and were at with a of The protocol with of using T. P. K. J. 2002; PubMed Scopus Google Scholar), with T. P. K. J. Biol. 2002; PubMed Scopus Google Scholar), and structure with P. Methods Biol. PubMed Scopus Google was and were from the chemical shift J. PubMed Scopus Google Scholar). the were with the in the for the structure the a of were from in the of which The zinc ion was in the of the form by a residue in the residue is from a of atoms that the to they can penetrate into the protein and with a of which the zinc The atoms of Cys ligands and of His-119 were to the metal ion through of and on the of does not of the ligands with to the zinc The with the function were to with D.A. T. H. R. C. J. Chem. PubMed Scopus Google Scholar). and were with of and respectively. The for the ion and the ligands were from for similar zinc in zinc proteins and L. I. S. J. Biol. 2002; PubMed Scopus Google Scholar, J. Chem. PubMed Scopus Google Scholar, L. Chem. Biol. 2003; PubMed Scopus Google Scholar). The of the was using the and R.A. R. J. 1996; PubMed Scopus Google Scholar, R.A. J. 1993; Google Scholar). was with the R. K. J. 1996; PubMed Scopus Google Scholar). Yeast Cox4 a residues in the C-terminal domain of bovine CoxVb a single Zn(II) Yeast Cox4 contains only three of the coordinating ligand exists in the position of the fourth Cys from the bovine CoxVb sequence (8Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (523) Google Scholar). yeast Cox4 is a an N-terminal truncate of yeast Cox4 was in E. coli. The residues that to the mitochondrial sequence R.A. J. Biol. Chem. Full Text PDF PubMed Google and an residues on the N-terminal side that in bovine CoxVb has an extended conformation The recombinant Cox4 was to be a protein containing Zn(II) in a Zn(II) Zn(II) is a metal with no Zn(II) is the coordination environment of yeast Cd(II) was into Cox4 using The ½ nuclear spin of 113Cd by The cadmium nucleus has its by and NMR of the complex a at based on a chemical shift position is of coordination by three and based on proteins (9Coleman J.E. Methods Enzymol. 1993; 227: 16-43Crossref PubMed Scopus (75) Google Scholar). atoms are provided by the three conserved cysteines in Yeast Cox4 contains four with eight residues the first with the remaining three in the C-terminal A Cox4 construct was that the C-terminal residues histidyl residues except of the recombinant C-terminal truncate of Zn(II). the with 113Cd(II) in a complex with NMR of the truncate a of The similar chemical shift of the C-terminal truncate that the C-terminal His residues not to Zn(II) His-119 as the donor to Zn(II) in the WT protein. The C-terminal sequence in Cox4 is to bind an Zn(II). Cox4 from E. coli in the of was with of a Cox4 truncate lacking the C-terminal residues only a single Zn(II) Zn(II) binding to the C-terminal results in Cox4 of Cox4 with results in a of the a similar titration with the C-terminal truncate to show of the C-terminal truncate complex was used to the coordination environment of the metal of the using a coordination environment with three at and either or or oxygen ligands at The and but which of these is can be by comparison of of the base that the are consistent with a a is as this for both and and are as a of histidine are not observed in does not His in the of from three of in the role of His-119 and the three conserved Cys residues in Zn(II) coordination and mutagenesis was Yeast transformed with a containing either WT COX4 or a gene was on for respiratory Cox4 mutants with Cys at the three conserved positions to on of the gene was on a or not the WT COX4 The of to the to not The Cys variants of Cox4 are proteins and fail to CcO fails to assemble in cells lacking Cox4 (12Glerum D.M. Tzagoloff A. FEBS Lett. 1997; 412: 410-414Crossref PubMed Scopus (38) Google Scholar). assembly is in cells with Cys mitochondria from cox4Δ cells containing Cox4 were to and for Cox2 was in the cells, a Cox2 was in WT cells not was on His-119 to its importance in Cox4 and were and were to provide a Zn(II) donor ligand. His Cox4 cells to on when but not when from a plasmid and as protein were CcO was observed in cells containing the mutants cells either the or Cox4 on when the mutants were on plasmids results are consistent with Zn(II) binding to the and Cox4 with the 113Cd NMR studies the C-terminal truncate lacking the C-terminal His residues WT Cox4 in respiratory not results His-119 as the fourth Zn(II) ligand in yeast on the Mutant the Cox4 was for metal we to use Co(II) electronic as a probe to the coordination environment. and Cox4 were to Co(II) for spectroscopy. The absorption in the visible between and from d-d transitions of electrons and information on the Co(II) coordination environment (10Bertini I. Luchinat C. Eichhorn G.L. Marzilli L.G. Advances in Inorganic Biochemistry. Elsevier Science Publishing Co., Inc., New York1984Google Scholar). The absorption of between and and is of a tetrahedral environment with coordination by three and ligand based on proteins The is consistent with three The absorption for the d-d transitions of is with the of the wild-type protein The wild-type construct has at and and a at the and were the fourth ligand in the substitution an oxygen donor that is a ligand with a The crystal the d-d in a shift of the electronic His-119 as the fourth ligand. of complex was to multidimensional NMR for structure His-119 was to be the fourth Zn(II) ligand by of the chemical shift difference of of the of a histidine residue for coordinated His residues for the or the two on the is or S. A. Biochemistry. 1995; PubMed Scopus Google Scholar). The chemical for and of His-119 are and consistent with the of a coordination between of His-119 and the Zn(II) for the of the in the on at show that His-119 is on The of at is indicative of an protein with The solution structure of as by using and has a to the structure of a of for the and a of for the The and of structure is in The fold contains β in of and of residues and ligands are located in two at of the with side in the of the protein core where the metal is located The zinc ion is, therefore, buried within the protein core with no that zinc a structural role the Cox4 has the same fold as that of the with The of of is, with the to that of The of a residue in Cox4 sequence to the of and as its insertion the of for the is not conserved in CoxVb but is by that is part of the hydrophobic in the protein core of thus a The zinc ion and the three conserved Cys ligands of the two are in the two The fourth in and His-119 in is located in loop in both but in a different loop in the two a different structural The loop in the zinc site in the region containing the region in is the the loop region containing in is to the Zn(II) but it is from in loop its conformation to the different sequence position of the metal ligand. with this is observed in the yeast and bovine primary of this loop The core of the is by a hydrophobic residues and which are conserved in the structure hydrophobic residues in and in are conserved on the of the residues form hydrophobic with and residues located at the terminus of the Cox1 subunit in the bovine CcO structure that these conserved hydrophobic residues play an important role in the Cox4-Cox1 in region in both and is at with the residues in the The Cox4 is of the two globular subunits of CcO the matrix side of the IM, the Cox4 is the only cofactor-containing subunit that is not directly part of the catalytic The role of Zn(II) binding in Cox4 has not been We show that CcO assembly and is on the of Cox4 and on Zn(II) coordination in Mutant proteins with of the three Cys residues are and fail to lead to an CcO complex as is the in cells of Cox4 (7McEwen J.E. Ko C. Kloeckner-Gruiissem B. Poyton R.O. J. Biol. Chem. 1986; 261: 11872-11879Abstract Full Text PDF PubMed Google Scholar, 12Glerum D.M. Tzagoloff A. FEBS Lett. 1997; 412: 410-414Crossref PubMed Scopus (38) Google Scholar). the structure of Cox4 is on Zn(II). The of is not Zn(II) binding to Cox4 is essential to the stability of Cox4 and subsequently Yeast Cox4 from the bovine ortholog in lacking of the Cys residues (8Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (523) Google Scholar). ligand exists in the position of the fourth conserved Cys from the bovine 113Cd NMR of Cox4 suggested that a single His residue to the Zn(II) coordination sphere. Because the chemical shift of the protein not deletion of the C-terminal residues containing of the His residues in the His-119 was as the Zn(II) ligand. was by the chemical shift of the His-119 and the solution structure The the is coordinated to Zn(II). The is the donor in proteins with structural Zn(II) The is in and it is to water or a protein the is not to a protein the that it is to or to a Cox1 Cys mutants in Cox4 are and Cox4 variants were when overexpressed. The function of CcO containing a Cox4 that Zn(II) coordination in this The Cox4 is with a Zn(II) and the Zn(II) is to of cox4Δ cells the but not the consistent with the as a ligand. The Cys mutants of Cox4 not be as the recombinant proteins were is not Cox4 has a role in to on the matrix side of the IM a CcO is to by ATP B. B. J. Biochem. PubMed Scopus Google Scholar, I. E. S. B. Biol. Chem. PubMed Scopus Google Scholar), and ATP is to on the matrix side of CcO, subunits Cox4 and as ATP of CcO is only observed in the eukaryotic that the peripheral subunits this ATP binding were the only subunit is which has a small matrix domain in to an IM subunit was as ATP site of CcO on the of studies R. G. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). yeast Cox4 is an ATP binding subunit, an NMR study was in the and of of in Cox4 was the C-terminal of Cox4 is of binding an Zn(II) and the complex is to The C-terminal of bovine CoxVb is in to the N-terminal sequence of Cox3 that is suggested to be important in proton through the J. Ferguson-Miller S. Mills D.A. Annu. Rev. Biochem. 2006; 75: 165-187Crossref PubMed Scopus (250) Google Scholar). Because Zn(II) binding is to the of proton A. A. P. 2002; PubMed Scopus Google Scholar), the exists that the C-terminal segment of Cox4 be involved in proton for proton of this Cox4 residues and show in chemical shift as a function of from a membrane in to ATP is of membrane are to B. E. S. I. B. PubMed Scopus Google Scholar). One mechanism to a membrane is a of The Cox4 be a through a mechanism analogous to the in E. coli J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, 2006; PubMed Scopus Google Scholar). is a protein that is in its as a protein. is to molecular The a Zn(II) and of ligands in Zn(II) designated as a zinc is to be a the zinc site in Cox4 is buried with no the of bovine CoxVb is the that Cox4 a for mitochondrial The two cysteinyl are buried and to be to of the yeast Cox4 CcO and the mitochondrial membrane will this of this work were at the which is by the of of and of and and the of for We the of Dr. Dr. and with

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