.J. Mol. Biol. (199%) 227. 583-585
Crystallization and Preliminary X-ray Analysis of the Methane Monooxygenase Hydroxylase Protein from Methylococcus capsulatus (Bath) Amy C. Rosenzweigl, Christin A. Frederick2 and Stephen J. Lippard’t ‘Department
of Chemistry, Massachusetts Institute Cambridge, ML4 02139, U.S.A.
of Technology
‘Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, Dana Farber Cancer Institute 44 Binney St, Boston, MA 02115. U.S.A. (Received
11 March
1992; accepted 20 May
1992)
I\/lethane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of met’hane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition xZBZy2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol. lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group Y21212,, with one dimer of relative molecular mass M,=252,000 in the asymmetric unit,. The unit cell dimensions are a = beyond 2.5 g resolution. 62.6 A, b = 1101 A, c = 333.5 8. The crystals diffract uniformly (Crystals of the related hydroxylase from Methylosin,us trichosporium OB3b have also been obtained. Keywords:
crystallization; methane monooxygenase; hydroxylase; Methylococcus cap~sulatus (Bath); Methylosinus trichosporium OB3b: X-ray crystallography
methanotrophic bacteria have the Obligate remarkable ability to utilize methane as a source of carbon for growth and energy (Hanson, 1980). The first, st,ep in this metabolic pathway is the oxidation of methane to methanol: C’H,+NADH+H+$02 +CH,OH+NAD+
+H,O.
(1)
Methanol is further oxidized to formaldehyde and carbon dioxide, and then assimilated via either the ribulose monophosphate pathway (type I methanotrophs) or the serine pathway (type IT methanotrophs: Anthony, 1982). The conversion of methane to methanol is catalyzed by the three proteins of the methane monooxygenase (MMOS) enzyme system that has been isolated and characterized from both the type I met,hanotroph Methylococcus capsulatus (Bath) and ttricz ttiffwwws in thtb region of t hrir tlinuc*fear iron cwtt ws. Although X-ray structures are avaifnhfr~ f’or hot h rnrta~idohetner~thritt (Stenkamp rot cd.. 19)x4: Sheriff 4 01.. I9X7) and the 132 sufmnit) of rif~otiuc~l~ot.ic~~~ rrduc~taw (Nordh~nd rf al.. 1990). muA less is known about t fw clet~aifrd geomptrirs of t,fw iron vows in met hanr monooxyygrnast~ and purple ;\ difirwtion analyses thr frydroxyf;~w c~ompowttts of’ MM0 frorrr I,oth N. cnp”“l”t’ms ( Bath) md .II. tri~lrosporiuttt OK3b. LV(b report htw t Iw si~vwssf’~~l wystallizat~ioti of tdi h~droxylasr prw tcitrs and prcfiminary S-ray diff&wtion itnalysis of thr protein from t)fw forniw sours. Tfte .W. cnpslrlntus (138th) hytlroxyla.se was purtied as previously dcwrib(d (DrLYit t rt (I/.. 1 WI ). anti tnatrriaf of)tainrd in this manner was usrtf for c*rystalfization trials. bt’ell over 1000 crystallization c*ontlitions werr scwened ty using the vapor diffw sion m&hod wit,h wide and systrtnatic vwiat~ion of t)uffers, salts. prwipitating agents, tetnperat~ure, f)H wid protein concwttration. Most) c~ryslaffizat.iort trials yirltlrd prwipitate. hut clustrrs of very tfiin f)fatw wwc obtained from solutions cwlit,aitting 6 mg in 25 m~-3-(,v-mor~hc,lillo) propaneprotein/ml suffonic~ acid (pH 74) 23 mw-lithium su1tnt.e. 23 mwammotiium acetatth. and Ho,, (Xv/v) ply-
__----~-
t+liylerw reservoir
glyc~)I 4000 ec~uilif~ralc~d i&t 1 (’ +gainst it tlyltw~ glycY~l writ aining ‘LO”,, (\I ;v) ply+ srparatd and ustvf for 1000. ‘I’htw fktrs were repeated seeding t~xperirnents following ;I St~i~Ild~lYl protocol (Stura k Wilson. 1990). Within tww uc,vks. t,hrl se& grew to Iargr plate-fikrb twtqgtiar with dim(wsionb crystals tttwxirtium OM mm x 02 mm x 0% trim. Thin I)latcb-fiktx carystals of’ thtl .U, tri~hospoliuttt OlUh h~tlroxylast~. purified using a prot~ovol in thr lit.erat,ure (Fo\: vf (I/.. 19X9). )\ilVt’ illSO hWt1 ohtainetf t)v using similar c*onclitiotis. Optimization of’ the’ c~rystalli~;~tio~r cwditions for the hycirosylasr from I his org,r;tl,istrt is utldw
way.
‘I’lw t hr,c,~,-tfilnrnsiott~tl st rtic?nrt~ of t ht. ft>~tfroxy~ law c~onif~onrnt of’ nitd harw Iric,tioo?c!.ir;r~ti~~s(’ is fJirst hi r1lv1 ural itnporiant f’c)r wvt~ral reRSo1lS. c~lIat.itc~tc~t.izatio~~ tatta
stucfira
of tflr
of thr
avti\;tt
tlltb. traininp grant (‘A 09ffP from t.ht‘ National (‘anc*c~r fnstittrtr (,\.(‘.R.).
References
(‘oIh>-. .I B f)afton. H. (197X). Resolution of the methams ~apsulafus (Bath) rttctnoox~eeiias~~ of’ M~thylococcus into thrrr cvmftonents. Biorhum. J. 171. 461-468. (‘oltty. ,I.. Wrline. I). I. & Dalton. H. (1977). Thp soluble tnrt,tiane monoox~genase of MethyZococor,s capsulntus ( ILtt h). 12io&/,/, .I. 165. 395-102. IkWitt. .I. 1:.. Brtttsrn. .J. G., R.osenzwt?ig, A. C‘.. I’ilkington, s.. (:rren, Hrdmait. IS.. J ., I’al)t’~‘filr~rnic,u. (:. C’.. f)alton. H.. Hod~s~~n, K. 0. & I,ippard. S. .I. (1!4!81). S-ray absorption. Miissbaurr. and Ef’R stttdivs of thr dittucalrar iron c*rnt,rr in the h?dr~os,vlasr cvmportent of methane mottoox~~grnasr. ./. .-I lllfl’. (‘hfrr,. ‘SOf.113. 9219-92X. f+Zric.sotr. A.. Hvclma.tt. f-lasr c*omponrttt of mrthanr IIIO~~~~IJX~~~~~~~SI~. J.
Bid.
(‘hem.
263.
105X3
Hanson, R. S. (1980). fG:olo~~ and tlivrrsit,y of AdlYl~n. .-I ppi. !Ifirrol/ir$. mrt~i~lotrofthic~ orpanisms. 26. 3 37. Lippard. S. ,J. (I 98X). Oxo-bridged pofyirott centers in biofog~ and c*hemistry. ilng~t~nnrl/+~ (‘l/~ntir~. Intrrnl. Edit. in English. 27. :M--361. Lit. K. E. Hr Lipftard. S. .I. (1991). Rtvlct.\ ftrof)erties of f >f’ nlrthatlr tt~tlroxyla~sr utm potlr’tlt the moiiooxvgenase from Methylococw wpsulnt/ts (Bath). :/. Hiol. (‘hum. 266. 16836--1X39 I,untl. .I. bz I)alton. H. (1985). Further charactrrizatiolt of thri FAI) and Fe2S, rrdox (*enters of c*onrponent (‘. t htx S:1 f)H : ac.c*t,ptor reduc+asr of’ I ttt3 solublr ~~rpsulnt~rs rnethatte motioox?‘prnase of Mrthyiocnwcrs (Kath). Eur. .I. Niorhrm. 147. “91 l!% J1atthrn.s. IS. \V. (196X). Solvent c*oittc,rtt of ltrotvin c,rgstals. ./. Mol. Hiol. 33. 1!1f -d!li. Notdlund. f’.. Sjiibrr#. R.-M. 6 Eklund, H. (1990). ‘rhr~,~-ditttr,ltniorIal strucaturr of tlrv frr~ radic~al SUtllW rt’dll(~taht’. of rif,orrlr~leotitir ftrotrilt
/ I,w/dotr) ~ 345. 59:s -598. C&c&. I,.. .Jr & True. A. K. (1990). tttatt~attesvoxo sites in Itiolo~y.
f)inuc~lr~ar irotl- ant1 C’ht n,. /‘/vfg. Inrjry.
38. 97 2W.
lM5B.
f4’0x. IS. C:.. T;‘rc,larttl. \\‘. .I.. I)rge. .J. B. & TAipsc~otnb. .J. I). (I W!,). Mvthatt(* tnottoctx~~~~~~ast‘ from Mrth!ylosincc.~ (‘/iflU. 264. .I. IZiol. f ) tc11,. I,-;fi/o.s~“‘rir,,,r I ooL’:1 I OOXI. FOG. I
Crystallization and Preliminary X-ray Analysis of the Methane Monooxygenase Hydroxylase Protein from Methylococcus capsulatus (Bath) Amy C. Rosenzweigl, Christin A. Frederick2 and Stephen J. Lippard’t ‘Department
of Chemistry, Massachusetts Institute Cambridge, ML4 02139, U.S.A.
of Technology
‘Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, Dana Farber Cancer Institute 44 Binney St, Boston, MA 02115. U.S.A. (Received
11 March
1992; accepted 20 May
1992)
I\/lethane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of met’hane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition xZBZy2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol. lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group Y21212,, with one dimer of relative molecular mass M,=252,000 in the asymmetric unit,. The unit cell dimensions are a = beyond 2.5 g resolution. 62.6 A, b = 1101 A, c = 333.5 8. The crystals diffract uniformly (Crystals of the related hydroxylase from Methylosin,us trichosporium OB3b have also been obtained. Keywords:
crystallization; methane monooxygenase; hydroxylase; Methylococcus cap~sulatus (Bath); Methylosinus trichosporium OB3b: X-ray crystallography
methanotrophic bacteria have the Obligate remarkable ability to utilize methane as a source of carbon for growth and energy (Hanson, 1980). The first, st,ep in this metabolic pathway is the oxidation of methane to methanol: C’H,+NADH+H+$02 +CH,OH+NAD+
+H,O.
(1)
Methanol is further oxidized to formaldehyde and carbon dioxide, and then assimilated via either the ribulose monophosphate pathway (type I methanotrophs) or the serine pathway (type IT methanotrophs: Anthony, 1982). The conversion of methane to methanol is catalyzed by the three proteins of the methane monooxygenase (MMOS) enzyme system that has been isolated and characterized from both the type I met,hanotroph Methylococcus capsulatus (Bath) and ttricz ttiffwwws in thtb region of t hrir tlinuc*fear iron cwtt ws. Although X-ray structures are avaifnhfr~ f’or hot h rnrta~idohetner~thritt (Stenkamp rot cd.. 19)x4: Sheriff 4 01.. I9X7) and the 132 sufmnit) of rif~otiuc~l~ot.ic~~~ rrduc~taw (Nordh~nd rf al.. 1990). muA less is known about t fw clet~aifrd geomptrirs of t,fw iron vows in met hanr monooxyygrnast~ and purple ;\ difirwtion analyses thr frydroxyf;~w c~ompowttts of’ MM0 frorrr I,oth N. cnp”“l”t’ms ( Bath) md .II. tri~lrosporiuttt OK3b. LV(b report htw t Iw si~vwssf’~~l wystallizat~ioti of tdi h~droxylasr prw tcitrs and prcfiminary S-ray diff&wtion itnalysis of thr protein from t)fw forniw sours. Tfte .W. cnpslrlntus (138th) hytlroxyla.se was purtied as previously dcwrib(d (DrLYit t rt (I/.. 1 WI ). anti tnatrriaf of)tainrd in this manner was usrtf for c*rystalfization trials. bt’ell over 1000 crystallization c*ontlitions werr scwened ty using the vapor diffw sion m&hod wit,h wide and systrtnatic vwiat~ion of t)uffers, salts. prwipitating agents, tetnperat~ure, f)H wid protein concwttration. Most) c~ryslaffizat.iort trials yirltlrd prwipitate. hut clustrrs of very tfiin f)fatw wwc obtained from solutions cwlit,aitting 6 mg in 25 m~-3-(,v-mor~hc,lillo) propaneprotein/ml suffonic~ acid (pH 74) 23 mw-lithium su1tnt.e. 23 mwammotiium acetatth. and Ho,, (Xv/v) ply-
__----~-
t+liylerw reservoir
glyc~)I 4000 ec~uilif~ralc~d i&t 1 (’ +gainst it tlyltw~ glycY~l writ aining ‘LO”,, (\I ;v) ply+ srparatd and ustvf for 1000. ‘I’htw fktrs were repeated seeding t~xperirnents following ;I St~i~Ild~lYl protocol (Stura k Wilson. 1990). Within tww uc,vks. t,hrl se& grew to Iargr plate-fikrb twtqgtiar with dim(wsionb crystals tttwxirtium OM mm x 02 mm x 0% trim. Thin I)latcb-fiktx carystals of’ thtl .U, tri~hospoliuttt OlUh h~tlroxylast~. purified using a prot~ovol in thr lit.erat,ure (Fo\: vf (I/.. 19X9). )\ilVt’ illSO hWt1 ohtainetf t)v using similar c*onclitiotis. Optimization of’ the’ c~rystalli~;~tio~r cwditions for the hycirosylasr from I his org,r;tl,istrt is utldw
way.
‘I’lw t hr,c,~,-tfilnrnsiott~tl st rtic?nrt~ of t ht. ft>~tfroxy~ law c~onif~onrnt of’ nitd harw Iric,tioo?c!.ir;r~ti~~s(’ is fJirst hi r1lv1 ural itnporiant f’c)r wvt~ral reRSo1lS. c~lIat.itc~tc~t.izatio~~ tatta
stucfira
of tflr
of thr
avti\;tt
tlltb. traininp grant (‘A 09ffP from t.ht‘ National (‘anc*c~r fnstittrtr (,\.(‘.R.).
References
(‘oIh>-. .I B f)afton. H. (197X). Resolution of the methams ~apsulafus (Bath) rttctnoox~eeiias~~ of’ M~thylococcus into thrrr cvmftonents. Biorhum. J. 171. 461-468. (‘oltty. ,I.. Wrline. I). I. & Dalton. H. (1977). Thp soluble tnrt,tiane monoox~genase of MethyZococor,s capsulntus ( ILtt h). 12io&/,/, .I. 165. 395-102. IkWitt. .I. 1:.. Brtttsrn. .J. G., R.osenzwt?ig, A. C‘.. I’ilkington, s.. (:rren, Hrdmait. IS.. J ., I’al)t’~‘filr~rnic,u. (:. C’.. f)alton. H.. Hod~s~~n, K. 0. & I,ippard. S. .I. (1!4!81). S-ray absorption. Miissbaurr. and Ef’R stttdivs of thr dittucalrar iron c*rnt,rr in the h?dr~os,vlasr cvmportent of methane mottoox~~grnasr. ./. .-I lllfl’. (‘hfrr,. ‘SOf.113. 9219-92X. f+Zric.sotr. A.. Hvclma.tt. f-lasr c*omponrttt of mrthanr IIIO~~~~IJX~~~~~~~SI~. J.
Bid.
(‘hem.
263.
105X3
Hanson, R. S. (1980). fG:olo~~ and tlivrrsit,y of AdlYl~n. .-I ppi. !Ifirrol/ir$. mrt~i~lotrofthic~ orpanisms. 26. 3 37. Lippard. S. ,J. (I 98X). Oxo-bridged pofyirott centers in biofog~ and c*hemistry. ilng~t~nnrl/+~ (‘l/~ntir~. Intrrnl. Edit. in English. 27. :M--361. Lit. K. E. Hr Lipftard. S. .I. (1991). Rtvlct.\ ftrof)erties of f >f’ nlrthatlr tt~tlroxyla~sr utm potlr’tlt the moiiooxvgenase from Methylococw wpsulnt/ts (Bath). :/. Hiol. (‘hum. 266. 16836--1X39 I,untl. .I. bz I)alton. H. (1985). Further charactrrizatiolt of thri FAI) and Fe2S, rrdox (*enters of c*onrponent (‘. t htx S:1 f)H : ac.c*t,ptor reduc+asr of’ I ttt3 solublr ~~rpsulnt~rs rnethatte motioox?‘prnase of Mrthyiocnwcrs (Kath). Eur. .I. Niorhrm. 147. “91 l!% J1atthrn.s. IS. \V. (196X). Solvent c*oittc,rtt of ltrotvin c,rgstals. ./. Mol. Hiol. 33. 1!1f -d!li. Notdlund. f’.. Sjiibrr#. R.-M. 6 Eklund, H. (1990). ‘rhr~,~-ditttr,ltniorIal strucaturr of tlrv frr~ radic~al SUtllW rt’dll(~taht’. of rif,orrlr~leotitir ftrotrilt
/ I,w/dotr) ~ 345. 59:s -598. C&c&. I,.. .Jr & True. A. K. (1990). tttatt~attesvoxo sites in Itiolo~y.
f)inuc~lr~ar irotl- ant1 C’ht n,. /‘/vfg. Inrjry.
38. 97 2W.
lM5B.
f4’0x. IS. C:.. T;‘rc,larttl. \\‘. .I.. I)rge. .J. B. & TAipsc~otnb. .J. I). (I W!,). Mvthatt(* tnottoctx~~~~~~ast‘ from Mrth!ylosincc.~ (‘/iflU. 264. .I. IZiol. f ) tc11,. I,-;fi/o.s~“‘rir,,,r I ooL’:1 I OOXI. FOG. I
