Volume 30% number 2, 127-129 0 1991 Fcdemlion aT Rurqwin l3ioelwmiall Sdclics
Scprcmbsr 1992
FEBS 114tlS 00145793/9265.00
The formate complex of the cytochrome bo quinol uxidase of Eschcrichia cdl exhibits a ‘g = 1%’EPR feature analogous to that uf ‘slow’qtachrome oxidase Melissa W, Calhoun’.
Robert B. Gcnnis” and John C. Salcrnob
The c~t~hron~c hu quinol oaid;lsc of kdwridh w/i is hamolcrpous in rqucncc and in s~ructurc to cyfochromc W, type cytochromc oxidvrc in subunn 1, which ccrntrlinr tkcruuilylic core The ryWchr~mc hcl enzyme forms II farnr~lc camplsx which cxhibik ‘x m I?’ und ‘p = 2.9’ EPR signals a~X band: similnr rigrrals how prcviourly been observed only in ussaciPGon with the‘rilaw’und fomlutc=ligirtcd SMCIofcytochromc orrldssc. Thcsc signuln urir from Wmsilian~ within inlc#xl spin mulliplcls idcntificd wilh the hon~loyo~~r hcmc-capper binucluar ccllalylic ccntcrs in both cnrjmcr. Quinol anidiwc:Cylorhramc
1. INTRODUCTION Mony preparations of cytochromc oxidw contain 3 significant proportion of the slow or resting st:ttc. u form of the cnzymc which rcucts sluggishly with uddcd ligands but which cun be converted CQo more rctictivc form by cyctillg with reducing cquivalcnts [i-3]. Slow enzyme is churxtcrizcd by a blue shifted Sorct band (ncur 417 nrn) at neutral rnd mildly acidic pH [2]. An unusuni clcctron prrr~magnctic rcson;Incc spectrum with u feature ncilr g = 12 at X 5rrnd, apparently arising from an cxcitcd doublet of iln integral spin system. is associated with the slow sfatc [2j. Detergent-solubilisd cytochromc oxidasc can be convcrtcd quuntittitivcly to the slow form by incubation at slightly acid pH. Formatc binding by cytochromc oxidesc products 11stiltc which mimics the slow cnoymc both in reactivity to addsd iigands and in spectroscopic features [4,§]. In pttrticular, binding of formtttc can fully elicit the g = 12 feature from prcpanitions in which the fast form othrrwisc prcdomintltcs. Sshoonovcr and Palmer [S] hrrvc recently dcmonstratcd that formate complcxcs resembling slow oxidasc can be formsd in intact mitochondrial mcmbrancs: this dcmcmstrtitcs
‘ES, .~~triri[l~ydroxymc~l~yl]mc~l~yl~2~~mincc~l~;lncsulfonic ucid; ZDTA, c~hylcncdinminc lclnxctic acid; EPR, ckt ron pnraniugnctic rcsontincc.
Ahhwirrriar~s:
Ci~~~cn~rf~rc trtltfrcss:J.C. Sulcrno, Bialoay Dcpurtmcnl and CcnLcr for Biochemistry and Biophysics, Rcnscclticr Polyicchnic Inslitutc, f’roy, NY 12180, USA,
hu; Parmilic complrx:
EPR: fZ. ruli
that formatc binding cun produce such states in cvcn the most intact prcpxurions. Cytochromc ho. one of two ubiquinol oxides from tiktrcrict~i:~ cuh. is closely rclutcd to cytoshromc au3 type tcrminnl oxidatics. Considcrablc scqurncc homoiogy exists [a] and both enzymes cant& hcmc-coppcr binusicnr catalytic sites [7,8]. The E. edi cnzymc has been much icss cxtcnsivcly studied than cytochromc (I+ While no evidence for a slow form of any quinoi oxidnsc exists. binding of formatc to sycochromc ho produce opticlll changes and slows the binding of other ligands. Analogous chungzs in the EPR spectra have not been reported; it is of interest to dctcrminc whether ,thc 15.cdi forms spcctroscopicaliy similar states. 2. MATERIALS AND METHODS E. co/i nr;rin RGl45. which contains clcva~cd
lcvclr of the cytochrome bo complex bul lucks lhc cyhxhromc bd complex, was grown in a rich buffcrcd medium ;IP dcrribcd previously [9,10]. Mcmbruncs wcrc prcparcd as dcscribcd by Lcmicux ct PI. [I I], Briefly, ccllr wcrc paused through II French prcuurc ccl1 gcncntiny hiyh rhcllr forcsr. and membranes were collcctcd by ultmccnWugation. Mcmbrunss wcrc w&cd once with 50 mM TES, pH 7,0,2 mM EDTA. and wcrc rcsuspcndcd in the same buffer et npproximatcly 40 mg prolcitiml. Cykxhromc oxidtisc was prcparcd by the mclhod 01’ Yu [ 121 with minor modifkations lnlroduccd 10 rcducc Ihccxposurc tocholicuund LO slightly r&c the pti during lhc purification, This rcsultsd in a rcduccd proponion of slow cnzymc in the preparation. nlthough -20% of the cnzymc as preparedWB~ still in the slow form by rpcclroscopic critcm [2]. Samples were prepared by incubaiing mcmbnncs or purified enzymes with formalc nt 50 mM or 20 mM. trimsfcrrinp lo 3 qzzrtt EPA *-_* r&c & rflptily ffekng in fald i*opcnlanr/ cyclohcxnnc (5: I v/v). Electron spin rcsonancc rpcc~r;l wcrc rccorclcd using ti Rrukcr ER300 spcclromctcr cquippcd with SW Oxford inrlru= mcnts cryos~ut c&cd with flowing liquid hcliu!n.
127
Volume 509, number 1
FEDS LETTERS
Scptcmbcr 1991
1
L
lOO0
A
2000 Mopetic
Fig. 1. EPR xpxm
Field
I
v,
3ow
1
do00
in Gaurr
of cymthromc ttq fcnninul oridrrrc akr
cubntion with 20 mM forma,
EPR condilicnr Fig. I.
in. wcrc as dsscribcd in
4. DISCUSSION Fig. I. EPR rpcarum
of mcmbrvncr from E w/i strain HGl45, amplified in cytochromc bo. A. As irolutcd with no uddilionr, B, Incubated with 20 mM formrlc; inrct, I’ = 12 rqion with gain incrcrrrcd by u fwlor of 4. EPR conditions wcrc: fcmpcr~turc. 13R: miwowwc power. 20 mW: microwwc frequency. 9.44 CiClz: moduh. lion smplitudc, 20 G: ficld sweep. 0.4 mT: ccntcr field pasition. 0.2 I mT: time caw;LnI. 163.W 111s:digital conversion intcrwl, 317.6tl ms: rcrolulion. 1024 flcld points; $wccp time, 33S.U I. I G = IO” I&I (73.
3. RESULTS Membranes of strain RGl45. prcpurcd us described above. were highly amplified in cytochromc bu and contuincd no cytochron~c bd. The electron paramagnctic rcsonuncc spectrum of the mcrnbruncs ns isolated is shown in Fig, 1; signals from the low spin ferric hems arc visible at g = 2.98 und g = 2.24. A signal at g = 6 arises from the high spin hcmc site. The rclativcly small sire of the g = 6 signal is due to the tight coupling bctwccn hcmc and copper in the binuclcar center; when both mctrlls arc oxidized. the coupling Icads to an EPR silent integral spin state. The g = 6 signals observed represent a minority population in which the copper is cithcr in the cuprous stats or absent. Thcsc signals arc greatly incrcascd at intcrmcdiatc potentials. reaching n maximum at about 300 mV. Fig. IB shows the effects of formatc rlddition on the EPR spectrum of cytochromc bo. An additional signal at g = 12 is visible. For purposes of comparison. the EPR spectrum of cytochromc q oxidasc in the prcscncc of 20 mM formatc is shown in Fig. 2. An almost identical signal at g = 12 is evident. In both Fig. IB and Fig. 2 n brortd signul at g = 2.9 is seen which is ovcrlapped with the gx feature of the low spin hcmc near g = 3, This has been attributed to the same state of the enzyme which gives rise to the g = I2 signal in cytochrome oxidnsc [ 131. it may rcprcscnt transitions bctwecn two other states in the integral spin multiplet which gives rise to the g = I2 signul, 128
The g = 12 signal in slow und formutc ligated cytochrome oxidaec arises from an excited doublet ol’ an intcgrul spin system. Scvcrul models for such L system huvc been proposed, The intcgrul spin system muy reprcscnt S = 2 ferry1 hcmc [ 14‘1S]. but ;I coupled system involving nntiferromagnctically coupled S = S/2 ferric hcmc to S = l/2 cupric copper to produce an S = 2 stntc has tliso bscn proposed [16,17], It hi\s been recently pointed out [ 181that thcg = 2.1 feature could arise from u different transition within the sumc intcgrul spin multiplct, but that some proposals (e.g. 1161)could not explain both transitions, The rclutivcly small (l-2 cm”) vulucs of the zero field splitting parrrmctcr D ncedcd to account for the signals has been presented us cvidcncc for the ferry1 hcmc model [14]. While it is true thut ferric hcmc D vulucs arc typically about five times this large and that rrurrha~c ferry1 iron complexes used as models have D values of the correct magnitude, it is worth pointing out that nonhcms high-spinfi’rrric D vnlucs arc also often smnll. The large D vr~lucsof high spin fcrrihcmcr reflect the gsomctry imposed by the tctropyrrolc. Recent evidence suggests that in the cytochromc oxidase formatc complcx hcmc CI!is high spin ferric and that the ground state isunS= quintet [t7]; no existing model accounts for all the date satisfactorily. Transitions bctwccn the excited state doublets arc likely to be first order forbidden, at lcust with the microwave mtlgnctic field component in its conventional orientation (perpendicular to the Zccman field). The g = 12 signals in both oxidascs urc very broad and less intcnsc than the g = 6. g = 3 and g = 2.24 signals of the ferric hcmes. In addition, it is necessary to USClower temperatures and/or higher microwuvc power to obtain rcnsonable signal to noise ratios for the g = I2 spccics at enzyme concentrations usunlly urrcd to study EPR signals from the ferric hcmc spccicu. It is probable that this is the rcnson thnr this signal has not been prcviousiy reported in the cytochromc bo system. Formatt ligation does not generally product ‘g = 12’ states in hcmc model systems; high-spin ferric hems is
Volume 309. number 2
FESS LE’ITERS
often observed. The observation of this unusual spcctroscopic feature almost unaltcrcd in the cytochromc ba system is further evidence for close structural homology between the two oxidasc systems. The scqucncc homology and structural similarity between the oxiduscs. particulurly within subunit 1. preserves enough common detail at the atomic lcvcl within the binuclcur catalytic site to allow the formation of such otherwise unique complexes. Superior ability to manipulate the cytochrome bu system should aid in our ability to choose bctwccn different models for unusual states such as the formutc lipted and resting enzymes. and to obtain as P result added insight into the structure of thrcutcllyticcorc of both the cytochromc CM+und cytochromc bo systems. Advuntagcs include rclsrtivcly easy gsnctic mtlnipulntion. ubility to obtain copper dcplctcd but hcmc sufficient cnzymc. und rcudy isotopic cnrichmcnt. Arkrta~~/crl~r~,rrrrrx: J.C.S. thanks
C.E. Cooper, W.J, Inylcdcw und P,R, Rich for valuable discussions. R,B,G, acknowlcdycs support from the D.O.E. (DEFG 01.87ER 13716).
REFERENCE§ [I] Anronini. E.. Brtinuri.
M.. Cobsimo, A.. Grccnwoad. C. curd Wilson. M. (19111)Prac. Natl. Awd. Sci. USA 78, II IS-‘I! tP,
sqmnber 1992
[2] Bnkcr, G.M. and Palmer, G. (19117)J.Biol. Chem. ?6?,595-604. [31 I”3;& L.J. wd Pirlmcr, G. (1985) J. Biol. Chcm. 261, 13031[St Moo&, Af.. Cooper, C.E. and Rich, P.R, (19911 Bioshim, Bio. phyr. Acrr 1059, 189-207. [51 $conovcr. J.R. rnd Palmer. G. (19991)Biochcmirtry 30.7541(61 Chc$uri. Y.. Lcmicux, L., Mill, J., Albcn. 1.0. and Gcnnh. R,B. (1990) Dioshim, Biophp. Aclu IOIB. 1%127. [I] hlcmo. J.C.. Bol&no, B, and Inglcdcw. W.J. (1989) FEBS La. 247, 101-107. [tl] Sulcrno. J.C., Balgiano, 5.. Pools, R.K.,Gcnnb, R.B. wl In&. dew. W.J. ( 1990) J. Biol. Chcm. 265, 4361-4368. [9] Au. DC-T. and Grnnir, R.B. (19117)J. Bactcriol. 161. 12%127. [IO] MinghaIL KC., Gorwitx. Y.C..GabricI. N.E.. HiII,J.J.. Brrnwsi, CA.. Chnn. S.1. and Cicnnb, R.D., rubmiltcd to Riochcmirtw. Lcmicur, L.J.. Calhoun, M,W., Thomas, J.W.. In&dew, $%J, and Gcnnis, R.B. (1992) J. I3iol. Chcm. 267. 2105-21 If, Yu. CA.. Yu. L. and King. T.E. (1975) J. Biol.Chcm. XI.
1383-1391.
Cooper. C.E., Moody. A.J.. Rich, P.R.. Wriyylcsworth.1.M. and loannidir. N. [I9911 Diochcm. SQS.Tmnr. 19.1595. Hit&en, W.R. il962j 5iochim. Biophyr. Acu iO.tl, 112-911. Dunham. W,R., Sands. R.H., Shtrw. R,W. und Bciucrt. M. (1983) Biarhim. Diaphyr. Acta 748, 73-85. Brudvig. G,W., kcvcnr.T.H,. Morse. R.H. undChtin.S.1. (1981) Biochcmislry Xl. 3912-3921. hrncr. Z.K., hbcock. G.T. und Dye. J.L. (1991) Oiuchcmisrry 30. 7597-7603. Coop. C.E. and S~lcrno, JC. (1992) J. Biol. Chsm. 267, 2X01115.
129
Scprcmbsr 1992
FEBS 114tlS 00145793/9265.00
The formate complex of the cytochrome bo quinol uxidase of Eschcrichia cdl exhibits a ‘g = 1%’EPR feature analogous to that uf ‘slow’qtachrome oxidase Melissa W, Calhoun’.
Robert B. Gcnnis” and John C. Salcrnob
The c~t~hron~c hu quinol oaid;lsc of kdwridh w/i is hamolcrpous in rqucncc and in s~ructurc to cyfochromc W, type cytochromc oxidvrc in subunn 1, which ccrntrlinr tkcruuilylic core The ryWchr~mc hcl enzyme forms II farnr~lc camplsx which cxhibik ‘x m I?’ und ‘p = 2.9’ EPR signals a~X band: similnr rigrrals how prcviourly been observed only in ussaciPGon with the‘rilaw’und fomlutc=ligirtcd SMCIofcytochromc orrldssc. Thcsc signuln urir from Wmsilian~ within inlc#xl spin mulliplcls idcntificd wilh the hon~loyo~~r hcmc-capper binucluar ccllalylic ccntcrs in both cnrjmcr. Quinol anidiwc:Cylorhramc
1. INTRODUCTION Mony preparations of cytochromc oxidw contain 3 significant proportion of the slow or resting st:ttc. u form of the cnzymc which rcucts sluggishly with uddcd ligands but which cun be converted CQo more rctictivc form by cyctillg with reducing cquivalcnts [i-3]. Slow enzyme is churxtcrizcd by a blue shifted Sorct band (ncur 417 nrn) at neutral rnd mildly acidic pH [2]. An unusuni clcctron prrr~magnctic rcson;Incc spectrum with u feature ncilr g = 12 at X 5rrnd, apparently arising from an cxcitcd doublet of iln integral spin system. is associated with the slow sfatc [2j. Detergent-solubilisd cytochromc oxidasc can be convcrtcd quuntittitivcly to the slow form by incubation at slightly acid pH. Formatc binding by cytochromc oxidesc products 11stiltc which mimics the slow cnoymc both in reactivity to addsd iigands and in spectroscopic features [4,§]. In pttrticular, binding of formtttc can fully elicit the g = 12 feature from prcpanitions in which the fast form othrrwisc prcdomintltcs. Sshoonovcr and Palmer [S] hrrvc recently dcmonstratcd that formate complcxcs resembling slow oxidasc can be formsd in intact mitochondrial mcmbrancs: this dcmcmstrtitcs
‘ES, .~~triri[l~ydroxymc~l~yl]mc~l~yl~2~~mincc~l~;lncsulfonic ucid; ZDTA, c~hylcncdinminc lclnxctic acid; EPR, ckt ron pnraniugnctic rcsontincc.
Ahhwirrriar~s:
Ci~~~cn~rf~rc trtltfrcss:J.C. Sulcrno, Bialoay Dcpurtmcnl and CcnLcr for Biochemistry and Biophysics, Rcnscclticr Polyicchnic Inslitutc, f’roy, NY 12180, USA,
hu; Parmilic complrx:
EPR: fZ. ruli
that formatc binding cun produce such states in cvcn the most intact prcpxurions. Cytochromc ho. one of two ubiquinol oxides from tiktrcrict~i:~ cuh. is closely rclutcd to cytoshromc au3 type tcrminnl oxidatics. Considcrablc scqurncc homoiogy exists [a] and both enzymes cant& hcmc-coppcr binusicnr catalytic sites [7,8]. The E. edi cnzymc has been much icss cxtcnsivcly studied than cytochromc (I+ While no evidence for a slow form of any quinoi oxidnsc exists. binding of formatc to sycochromc ho produce opticlll changes and slows the binding of other ligands. Analogous chungzs in the EPR spectra have not been reported; it is of interest to dctcrminc whether ,thc 15.cdi forms spcctroscopicaliy similar states. 2. MATERIALS AND METHODS E. co/i nr;rin RGl45. which contains clcva~cd
lcvclr of the cytochrome bo complex bul lucks lhc cyhxhromc bd complex, was grown in a rich buffcrcd medium ;IP dcrribcd previously [9,10]. Mcmbruncs wcrc prcparcd as dcscribcd by Lcmicux ct PI. [I I], Briefly, ccllr wcrc paused through II French prcuurc ccl1 gcncntiny hiyh rhcllr forcsr. and membranes were collcctcd by ultmccnWugation. Mcmbrunss wcrc w&cd once with 50 mM TES, pH 7,0,2 mM EDTA. and wcrc rcsuspcndcd in the same buffer et npproximatcly 40 mg prolcitiml. Cykxhromc oxidtisc was prcparcd by the mclhod 01’ Yu [ 121 with minor modifkations lnlroduccd 10 rcducc Ihccxposurc tocholicuund LO slightly r&c the pti during lhc purification, This rcsultsd in a rcduccd proponion of slow cnzymc in the preparation. nlthough -20% of the cnzymc as preparedWB~ still in the slow form by rpcclroscopic critcm [2]. Samples were prepared by incubaiing mcmbnncs or purified enzymes with formalc nt 50 mM or 20 mM. trimsfcrrinp lo 3 qzzrtt EPA *-_* r&c & rflptily ffekng in fald i*opcnlanr/ cyclohcxnnc (5: I v/v). Electron spin rcsonancc rpcc~r;l wcrc rccorclcd using ti Rrukcr ER300 spcclromctcr cquippcd with SW Oxford inrlru= mcnts cryos~ut c&cd with flowing liquid hcliu!n.
127
Volume 509, number 1
FEDS LETTERS
Scptcmbcr 1991
1
L
lOO0
A
2000 Mopetic
Fig. 1. EPR xpxm
Field
I
v,
3ow
1
do00
in Gaurr
of cymthromc ttq fcnninul oridrrrc akr
cubntion with 20 mM forma,
EPR condilicnr Fig. I.
in. wcrc as dsscribcd in
4. DISCUSSION Fig. I. EPR rpcarum
of mcmbrvncr from E w/i strain HGl45, amplified in cytochromc bo. A. As irolutcd with no uddilionr, B, Incubated with 20 mM formrlc; inrct, I’ = 12 rqion with gain incrcrrrcd by u fwlor of 4. EPR conditions wcrc: fcmpcr~turc. 13R: miwowwc power. 20 mW: microwwc frequency. 9.44 CiClz: moduh. lion smplitudc, 20 G: ficld sweep. 0.4 mT: ccntcr field pasition. 0.2 I mT: time caw;LnI. 163.W 111s:digital conversion intcrwl, 317.6tl ms: rcrolulion. 1024 flcld points; $wccp time, 33S.U I. I G = IO” I&I (73.
3. RESULTS Membranes of strain RGl45. prcpurcd us described above. were highly amplified in cytochromc bu and contuincd no cytochron~c bd. The electron paramagnctic rcsonuncc spectrum of the mcrnbruncs ns isolated is shown in Fig, 1; signals from the low spin ferric hems arc visible at g = 2.98 und g = 2.24. A signal at g = 6 arises from the high spin hcmc site. The rclativcly small sire of the g = 6 signal is due to the tight coupling bctwccn hcmc and copper in the binuclcar center; when both mctrlls arc oxidized. the coupling Icads to an EPR silent integral spin state. The g = 6 signals observed represent a minority population in which the copper is cithcr in the cuprous stats or absent. Thcsc signals arc greatly incrcascd at intcrmcdiatc potentials. reaching n maximum at about 300 mV. Fig. IB shows the effects of formatc rlddition on the EPR spectrum of cytochromc bo. An additional signal at g = 12 is visible. For purposes of comparison. the EPR spectrum of cytochromc q oxidasc in the prcscncc of 20 mM formatc is shown in Fig. 2. An almost identical signal at g = 12 is evident. In both Fig. IB and Fig. 2 n brortd signul at g = 2.9 is seen which is ovcrlapped with the gx feature of the low spin hcmc near g = 3, This has been attributed to the same state of the enzyme which gives rise to the g = I2 signal in cytochrome oxidnsc [ 131. it may rcprcscnt transitions bctwecn two other states in the integral spin multiplet which gives rise to the g = I2 signul, 128
The g = 12 signal in slow und formutc ligated cytochrome oxidaec arises from an excited doublet ol’ an intcgrul spin system. Scvcrul models for such L system huvc been proposed, The intcgrul spin system muy reprcscnt S = 2 ferry1 hcmc [ 14‘1S]. but ;I coupled system involving nntiferromagnctically coupled S = S/2 ferric hcmc to S = l/2 cupric copper to produce an S = 2 stntc has tliso bscn proposed [16,17], It hi\s been recently pointed out [ 181that thcg = 2.1 feature could arise from u different transition within the sumc intcgrul spin multiplct, but that some proposals (e.g. 1161)could not explain both transitions, The rclutivcly small (l-2 cm”) vulucs of the zero field splitting parrrmctcr D ncedcd to account for the signals has been presented us cvidcncc for the ferry1 hcmc model [14]. While it is true thut ferric hcmc D vulucs arc typically about five times this large and that rrurrha~c ferry1 iron complexes used as models have D values of the correct magnitude, it is worth pointing out that nonhcms high-spinfi’rrric D vnlucs arc also often smnll. The large D vr~lucsof high spin fcrrihcmcr reflect the gsomctry imposed by the tctropyrrolc. Recent evidence suggests that in the cytochromc oxidase formatc complcx hcmc CI!is high spin ferric and that the ground state isunS= quintet [t7]; no existing model accounts for all the date satisfactorily. Transitions bctwccn the excited state doublets arc likely to be first order forbidden, at lcust with the microwave mtlgnctic field component in its conventional orientation (perpendicular to the Zccman field). The g = 12 signals in both oxidascs urc very broad and less intcnsc than the g = 6. g = 3 and g = 2.24 signals of the ferric hcmes. In addition, it is necessary to USClower temperatures and/or higher microwuvc power to obtain rcnsonable signal to noise ratios for the g = I2 spccics at enzyme concentrations usunlly urrcd to study EPR signals from the ferric hcmc spccicu. It is probable that this is the rcnson thnr this signal has not been prcviousiy reported in the cytochromc bo system. Formatt ligation does not generally product ‘g = 12’ states in hcmc model systems; high-spin ferric hems is
Volume 309. number 2
FESS LE’ITERS
often observed. The observation of this unusual spcctroscopic feature almost unaltcrcd in the cytochromc ba system is further evidence for close structural homology between the two oxidasc systems. The scqucncc homology and structural similarity between the oxiduscs. particulurly within subunit 1. preserves enough common detail at the atomic lcvcl within the binuclcur catalytic site to allow the formation of such otherwise unique complexes. Superior ability to manipulate the cytochrome bu system should aid in our ability to choose bctwccn different models for unusual states such as the formutc lipted and resting enzymes. and to obtain as P result added insight into the structure of thrcutcllyticcorc of both the cytochromc CM+und cytochromc bo systems. Advuntagcs include rclsrtivcly easy gsnctic mtlnipulntion. ubility to obtain copper dcplctcd but hcmc sufficient cnzymc. und rcudy isotopic cnrichmcnt. Arkrta~~/crl~r~,rrrrrx: J.C.S. thanks
C.E. Cooper, W.J, Inylcdcw und P,R, Rich for valuable discussions. R,B,G, acknowlcdycs support from the D.O.E. (DEFG 01.87ER 13716).
REFERENCE§ [I] Anronini. E.. Brtinuri.
M.. Cobsimo, A.. Grccnwoad. C. curd Wilson. M. (19111)Prac. Natl. Awd. Sci. USA 78, II IS-‘I! tP,
sqmnber 1992
[2] Bnkcr, G.M. and Palmer, G. (19117)J.Biol. Chem. ?6?,595-604. [31 I”3;& L.J. wd Pirlmcr, G. (1985) J. Biol. Chcm. 261, 13031[St Moo&, Af.. Cooper, C.E. and Rich, P.R, (19911 Bioshim, Bio. phyr. Acrr 1059, 189-207. [51 $conovcr. J.R. rnd Palmer. G. (19991)Biochcmirtry 30.7541(61 Chc$uri. Y.. Lcmicux, L., Mill, J., Albcn. 1.0. and Gcnnh. R,B. (1990) Dioshim, Biophp. Aclu IOIB. 1%127. [I] hlcmo. J.C.. Bol&no, B, and Inglcdcw. W.J. (1989) FEBS La. 247, 101-107. [tl] Sulcrno. J.C., Balgiano, 5.. Pools, R.K.,Gcnnb, R.B. wl In&. dew. W.J. ( 1990) J. Biol. Chcm. 265, 4361-4368. [9] Au. DC-T. and Grnnir, R.B. (19117)J. Bactcriol. 161. 12%127. [IO] MinghaIL KC., Gorwitx. Y.C..GabricI. N.E.. HiII,J.J.. Brrnwsi, CA.. Chnn. S.1. and Cicnnb, R.D., rubmiltcd to Riochcmirtw. Lcmicur, L.J.. Calhoun, M,W., Thomas, J.W.. In&dew, $%J, and Gcnnis, R.B. (1992) J. I3iol. Chcm. 267. 2105-21 If, Yu. CA.. Yu. L. and King. T.E. (1975) J. Biol.Chcm. XI.
1383-1391.
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