Characterization of YpmQ, an Accessory Protein Required for the Expression of Cytochrome c Oxidase in Bacillus subtilis

A search of the Bacillus subtilisgenome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase.B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, CuA and CuB, and a menaquinol oxidase that has only CuB. Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the ΔypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the CuA site of cytochrome coxidase. A search of the Bacillus subtilisgenome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase.B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, CuA and CuB, and a menaquinol oxidase that has only CuB. Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the ΔypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the CuA site of cytochrome coxidase. neomycin polymerase chain reaction polyvinylidene difluoride N,N,N′,N′-tetramethyl-p-phenylenediamine polyacrylamide gel electrophoresis base pair Cytochrome oxidases are integral membrane protein complexes that catalyze the reduction of oxygen to water and capture some of the redox free energy of this reaction as a transmembrane electrochemical gradient. The key structural features that are shared among all members of this family of enzymes are found in its largest subunit, subunit I. Subunit I is an integral membrane protein with 12–14 membrane-spanning helical segments that provide binding sites for two heme A moieties, known as cytochrome a and cytochromea 3, and one copper center, CuB. Cytochrome a 3 sits in close proximity to CuB, and together they form a binuclear site that is responsible for binding oxygen and its partially reduced states, which arise transiently in the course of catalysis. Cytochrome ais a low spin heme that functions to deliver electrons to cytochromea 3-CuB (1Michel H. Behr J. Harrenga A. Kannt A. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 329-356Crossref PubMed Scopus (391) Google Scholar). The nature of the electron input site reflects a division in the cytochrome oxidase family of enzymes into two groups. In cytochromec oxidases, such as the enzyme found in the mitochondrial inner membrane of eukaryotes, reducing equivalents are delivered from the soluble protein ferrocytochrome c. The cytochromec interaction site on the oxidase is predominantly defined by subunit II (2Millett F. de Jong C. Paulson L. Capaldi R.A. Biochemistry. 1983; 22: 546-552Crossref PubMed Scopus (127) Google Scholar, 3Taha T.S. Ferguson-Miller S. Biochemistry. 1992; 31: 9090-9097Crossref PubMed Scopus (26) Google Scholar). Subunit II has two transmembrane helices that anchor a solvent-exposed domain, which provides the inner sphere ligands for the dinuclear CuA center. Although there is no direct structural information on a cytochromec-cytochrome c oxidase complex, kinetic (4Hill B.C. J. Biol. Chem. 1991; 266: 2219-2226Abstract Full Text PDF PubMed Google Scholar, 5Hill B.C. Biochemistry. 1996; 35: 6136-6143Crossref PubMed Scopus (18) Google Scholar), mutagenic (6Zhen Y. Hoganson C.W. Babcock G.T. Ferguson-Miller S. J. Biol. Chem. 1999; 274: 38032-38041Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 7Wang K. Zhen Y. Sadoski R. Grinnell S. Geren L. Ferguson-Miller S. Durham B. Millett F. J. Biol. Chem. 1999; 274: 38042-38050Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and modeling (8Roberts V.A. Pique M.E. J. Biol. Chem. 1999; 274: 38051-38060Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) studies indicate that cytochromec binds at a site near the CuA center to allow for efficient electron transfer from cytochrome c to CuA. Thus, electrons enter cytochrome c oxidase via CuA and are transferred to the cytochromea 3-CuB center through cytochromea (9Hill B.C. J. Bioenerg. Biomembr. 1993; 25: 115-120Crossref PubMed Scopus (46) Google Scholar). The second group within the cytochrome oxidase family is the quinol oxidases, which receive reducing equivalents from a lipid-soluble quinol. The best known of this group is the ubiquinol oxidase from Escherichia coli (10Gennis R.B. Biochem. Soc. Trans. 1993; 21: 992-998Crossref PubMed Scopus (6) Google Scholar). Even though the quinol oxidases do not oxidize cytochrome c they do have a subunit II that has overall homology with the subunit II of the cytochromec oxidases. The major difference in subunit II of the quinol oxidase is the lack of the amino acid ligands for the CuA center. The lack of CuA accounts for the lack of reactivity of the quinol oxidases with cytochromec. Copper is an element that is used in proteins to fulfill specific catalytic and structural roles. However, copper is also a potential danger in biological systems due to its ability to catalyze oxidative damage of many cellular components. A new class of proteins, metallochaperones, have been identified that mediate the incorporation of copper into a variety of specific binding sites (11Askwith C. Kaplan J. Trends Biochem. Sci. 1998; 23: 135-138Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). As outlined above, the integral membrane enzyme cytochrome c oxidase has two biochemically and physically distinct copper centers. The CuA center is composed of two copper ions that are held by a set of amino acid ligands such that the coppers are within bonding distance from one another (12Blackburn N.J. Barr M.E. Woodruff W.H. van der Oost J. de Vries S. Biochemistry. 1994; 33: 10401-10407Crossref PubMed Scopus (147) Google Scholar). The second copper center of cytochromec oxidases is known as CuB and is physically associated with cytochrome a 3. CuAis contained in the extra-membranous domain of subunit II, whereas CuB is found in the membrane-embedded domain of subunit I (13Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1993) Google Scholar, 14Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1303) Google Scholar). There is much known about the role of these copper centers in the catalytic cycle of the enzyme, but relatively little is known about the mechanism of their assembly. A number of protein factors have been proposed to have a role in the assembly of complex integral membrane proteins such as cytochromec oxidase (15Schulze M. Rodel G. Mol. Gen. Genet. 1988; 211: 492-498Crossref PubMed Scopus (101) Google Scholar). In the last few years some of these assembly factors have been more specifically defined. A pathway for the import of copper into and its assembly into the copper centers of cytochrome c oxidase has been proposed from studies in Science. PubMed Scopus Google Scholar). A pair of copper A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) and S. 1996; PubMed Scopus Google Scholar), have been found in the membrane that are responsible for the of Copper is to a set of soluble binding proteins, one of which is binds copper in the and to the mitochondrial by an as protein which is an integral of the inner mitochondrial has been in copper delivery to cytochrome c oxidase in are by of Sco1 A. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). copper to cytochrome c or to is not Sco1 a direct role in the transfer of copper to cytochrome c oxidase to the transfer of copper from to cytochrome coxidase. the role of Sco1 as a of copper in J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to the specific for the assembly of the CuA and CuB centers in cytochrome c to at their assembly in the chain of the Bacillus B. subtilis expresses a cytochrome c oxidase the mitochondrial enzyme, has CuA and CuB centers within of subunit II and subunit The cytochrome c oxidase of B. subtilis has the added of a cytochrome to its subunit II, and this among the 3 of the cytochrome oxidase In B. subtilis expresses a menaquinol oxidase that cytochrome cytochrome a 3, and CuB within a subunit I that is to subunit I of 3. The menaquinol oxidase have a CuA center, but subunit II of menaquinol oxidase is to subunit II of 3. The amino used to form the CuA site are in the of B. subtilis menaquinol oxidase M. F. G. A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). The of these two members of the cytochrome oxidase family with distinct copper B. subtilis a for the of copper center assembly. A search of the B. subtilis a potential of the mitochondrial protein Sco1 but not a of that of the expression in cytochrome c In the expression of menaquinol oxidase is in the We that the from the by the growth medium with copper or by ypmQ from a plasmid. We have to two conserved and one histidine within the of YpmQ that a role for these as potential is proposed that YpmQ is involved in the assembly of the but is not involved in the assembly of the B. subtilis and Escherichia coli used are in I. E. coli on medium at also contained B. subtilis on medium L. J. B.C. Biochem. J. 1995; PubMed Scopus (26) Google Scholar) or base at for and growth as and and from and F. PubMed Scopus Google S. G. Mol. Gen. Genet. PubMed Scopus Google M. R. 1994; PubMed Scopus Google ypmQ, ypmQ, subtilis Cytochrome c and Cytochrome c of Bacillus of of the coli J. S. E. M. PubMed Scopus Google F. M. M. B. L. I. Mol. 1993; PubMed Scopus Google and are in ypmQ into in with whereas in the in a new and are in ypmQ into in with whereas in the The ypmQ by from B. subtilis by and which contained and and polymerase as The into F. PubMed Scopus Google Scholar) and used to E. coli to The of with with polymerase and and into S. G. Mol. Gen. Genet. PubMed Scopus Google Scholar) that with that used to E. coli to and found to to the F. I. G. A. S. R. L. A. M. S. S. B. A. Nature. PubMed Scopus Google Scholar) with the of a of to in a from to at in the amino acid has been by the with contained ypmQ in to the in even though have from the has been that with that in are in B. In to ypmQ in the as used in a 1996; PubMed Scopus Google Scholar) to contained ypmQ in the as but only in E. coli or coli strain F. M. M. B. L. I. 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The is the efficient as has for of this is in which with the of cytochromec oxidase M. T. M. M. J. Biochem. 1991; PubMed Scopus Google Scholar). YpmQ is to have a of from I. J. Mol. Biol. 1998; PubMed Scopus Google Scholar), to a that is a protein. of the transmembrane that also the of amino that the of J. 1995; PubMed Scopus Google Scholar). The of the of YpmQ and Sco1 and identifies a and a histidine in YpmQ that are conserved with The ypmQ from the of B. subtilis to A used to of the ypmQ in to this which not in B. of B. subtilis with with ypmQ on the to the is and A on the of the that from by cytochrome used to B. subtilis for the of cytochrome c oxidase activity J. PubMed Google Scholar). is to that is a electron to menaquinol the heme oxidase in membrane of B. subtilis B.C. L. J. Biochem. Biophys. 1993; PubMed Scopus Google Scholar). the that in this with a number of B. subtilis A in wild type B. subtilis and is in strain in which the expression of cytochromec oxidase is by of the cytochrome c oxidase and The strain of B. subtilis in which ypmQ is is also to oxidize ypmQ is to strain on the ability to oxidize is In to that the ability of to oxidize is not due to an expressed ypmQ in a in cytochrome c oxidase. is to oxidize as this activity is to the expression of cytochromec oxidase. in is the of the of a on the expression of ypmQ The is to the wild type protein in its ability to complement ΔypmQ ypmQ identified by its to Sco1 from and Sco1 is in copper delivery to cytochromec to the of cytochrome c oxidase that when ypmQ is by the growth medium with We membrane from wild type B. in which ypmQ is and in which the ypmQ deletion is by expression from a plasmid. of low copper and are with in medium copper 3 and medium used for of wild type B. subtilis copper in a In the low copper growth copper from the and the medium with of the copper In copper the low copper medium with which in not to the growth of wild type B. subtilis or strain The for membrane from wild and with low copper are with with copper In this the at is a of the enzyme activity and the amount of a 3. In the level of the to of the and which a level of reduction the is at which the to We have that of B. subtilis two that are reduced in this cytochromec oxidase and menaquinol oxidase B.C. L. J. Biochem. Biophys. 1993; PubMed Scopus Google Scholar). more of the from is by cytochromec oxidase. The cytochrome c oxidase activity of the wild type extract is similar to that B.C. L. J. Biochem. Biophys. 1993; PubMed Scopus Google Scholar). The activity of on low copper is reduced to the wild type extract activity of is due to of the menaquinol oxidase. The activity of is about of the wild type The of copper to the growth medium of the activity more the strain with low which to a level about that with the wild type of membrane for cytochrome 3 subunit on to and with 3 is a from wild type B. subtilis of is from with low copper of 3 is with low copper of and is with copper of 3 cytochrome c oxidase and subunit II of membrane from subtilis and copper of subunit low low low in a new also the cytochrome for the In with low copper the 3 in the extract is reduced to about that in the wild type The 3 is due to the of menaquinol oxidase. is in with of the cytochrome c oxidase and menaquinol oxidase in wild type B.C. L. J. Biochem. Biophys. 1993; PubMed Scopus Google Scholar) and the of cytochrome c oxidase in the growth medium is with copper the cytochrome 3 of is to that of wild In strain the level of cytochrome 3 a to of the wild type cytochrome are with the which that to a the cytochrome c oxidase these has close to the We have also found that the menaquinol oxidase activity is the in these membrane that the lack of YpmQ not its assembly. In with low copper the and activity of the cytochrome c oxidase are the of an in which to a form of the 3 complex used to the of the enzyme in this from wild type B. strain and from on an gel and transferred to for The two major I and II, are in the wild type and are in strain with low to 3 has reactivity with subunit II, and the of the subunit II for the in the amount of membrane protein in of The level of subunit II is more wild type in and to more of wild type in with low In with copper the subunit II level is of the with wild The for subunit II are with the cytochrome and activity of these A key of ypmQ is a set of conserved that are proposed to function in binding In the cysteines at and and histidine at are conserved when with the of Sco1 from K. I. S. J. G. E. R. Kaplan S. E. M. S. Genet. 1999; 23: PubMed Scopus Google Scholar). We expressed a with amino and replaced by and together and to and are not to oxidize and have a to the cytochrome c oxidase strain the cytochrome of subtilis strain wild type YpmQ to a strain an mutant of YpmQ and The strain wild type YpmQ has an at that is a of from the two heme oxidases In the strain an mutant of YpmQ has an at which is of the menaquinol oxidase. the difference between the membrane extract of the wild type strain the mutant strain a in cytochrome c oxidase The difference has a set of at and due to the cytochrome c domain of subunit II and at and due to a 3, which are of B. subtilis cytochrome c oxidase. There is also a difference in the level of cytochrome in the two as by the at in the difference The lack of cytochrome c oxidase in the of YpmQ due to expression or of the mutant forms of this have of the histidine on of these and used an to A of membrane by that YpmQ and mutant YpmQ proteins are at similar in these the are expressed to a similar level as wild type and are in the is that the to or the histidine to alanine have the expression and of these mutant YpmQ The of deletion on menaquinol oxidase function also by growth and has been that a in menaquinol oxidase to a and to B. subtilis M. F. G. A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, G. M. S. A. Biophys. 1995; PubMed Scopus Google Scholar). We have that a in which the menaquinol oxidase is is to whereas the wild type strain and to in the of We have also that has a that is similar to the wild type strain when are with the for not kinetic and growth the that deletion not the expression of menaquinol oxidase. of the assembly of copper centers in a number of proteins has in the of specific proteins that aid the assembly In some transfer of copper from the assembly protein to an has been and these proteins are identified as copper new class of proteins functions to deliver copper and the potential that arise from redox by free the copper center in in and is with the aid of the copper J. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the of mitochondrial cytochrome c oxidase has been that the protein is responsible for copper from the to the of the is into cytochrome c oxidase J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that are in are to on but by of copper to the growth medium A. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) or by of the inner mitochondrial membrane Sco1 A. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In by of copper to the growth Thus, the for copper delivery to cytochromec oxidase has copper in the through the mitochondrial membrane and copper to the A. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). copper is from to cytochrome c oxidase or with the of is not In B. subtilis two members of the cytochrome oxidase family are expressed that have copper The cytochrome c oxidase complex is similar to the mitochondrial enzyme in CuA and The menaquinol oxidase not have CuA but have the redox CuB. the expression of the Sco1 homolog, is the major and activity of 3 are the of the menaquinol oxidase. partially when copper is added to the growth medium and more when YpmQ is expressed from a plasmid. to suggest that YpmQ is involved in the assembly of CuA but is not involved with CuB assembly. suggest that the that the role of in is to deliver copper to the mitochondrial but Sco1 copper incorporation into cytochrome c oxidase. B. subtilis a of of its However, is to the direct role of these proteins in copper delivery to cytochrome c and the of an protein to In is that the to YpmQ in a of the cytochrome c oxidase on the quinol oxidase from E. coli that of copper from the growth medium an enzyme that has the low and spin heme centers but CuB PubMed Scopus Google Scholar). and L. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) similar in cytochrome c oxidase of an assembly is Deletion in the expression of a cytochrome complex that has all the redox centers of the enzyme, but CuB. on this and that on the E. coli that is to a enzyme and that this in the assembly of the oxidase In in do not a cytochromec oxidase complex that the CuA center, and this that CuA assembly at an and is for or for the overall of the complex in In a A. G. A. A. K. Rodel G. Genet. 1999; 35: PubMed Scopus (80) Google Scholar) on of Sco1 from two conserved which have in to and these to However, as the of this they that the mutant forms of Sco1 in some and that this for the However, that mutant and wild type of YpmQ are expressed and are all found in the We that the two conserved in the conserved histidine a direct role in the function of YpmQ is a of the proteins from and the cysteines and histidine are also critical to their function M. R. 1994; PubMed Scopus Google Scholar). We have wild type YpmQ as as in which of the copper has been We of these proteins and their to the role proposed for these in copper The copper has homology to its R.A. Struct. Biol. 1999; PubMed Scopus Google Scholar). In that to an by a amino acid M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The structural between and is proposed to mediate copper exchange via complex In the of cytochrome the copper has been to two copper ions C. Biochemistry. 1998; PubMed Scopus Google Scholar). of that one of its is a dinuclear copper center, the of the two sites is Sco1 and YpmQ homology with a amino of the CuA domain of cytochrome that is the pair of of the sites A. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). structural between the CuA site and its and YpmQ or do not a mechanism such as that proposed for the the that YpmQ is a of the protein Sco1 and that a role in the assembly of cytochrome c oxidase in B. Deletion the expression of cytochrome but not of menaquinol oxidase. propose that the protein by ypmQ is involved specifically in the assembly of CuA but is not for the assembly of CuB. a of the structural features of the CuA and CuB centers. CuA is in an domain about from the of the whereas CuB is to histidine contained within a transmembrane of subunit I and is about from the of the protein (13Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1993) Google Scholar, 14Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1995; 269: 1069-1074Crossref PubMed Scopus (1303) Google Scholar).

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