Crystallization of beef heart cytochrome c oxidase

JOllrnal of Crystal Growth 110 (1991) 247-251 N( ,rth-Holland

247

Crystallization of beef heart cytochrome c oxidase Sltinya Y o s h i k a w a , K y o k o S h i n z a w a Ba ~lc Research Laboratoo', Hlrnejt Institute of Technology, 2167 Shosha Htmefl 671-22, Japan

T,)mitake Tsukahara, T o s h i o A b e D~ 7artment of Industrial Chemtsto; Tottort Untt)er~lty, Tottorl 680, Japan

ard VVlnslow S. C a u g h e y De mrtment of Biochemistry, Colorado State UmverstO; Fort Collins, Colorado 80523, USA

The three-dimensional structure of cytochrome c oxadase, a complex (multlmetal, mulnsubunlt) membrane protein is critical to elu :~datlon of the mechamsm of the enzyn'uc reactions and their control Our recent developments in the crystalhzatlon of the enzyme 1so ated from beef hearts are presented The crystals appeared more readily at h~gher protein concentration, lower runic strength, hig her detergent concentration (Bnj-35) and lower temperature Large crystals were obtained by changing one of these parameters to the crystalhzatlon point as slowly as possible, keeping the other parameters constant. Increasing the detergent concentration was the mc st successful method, producing green crystals of the resting oxidized form as hexagonal blpyrarmds with typical dlmensmns of 0.6 nmt The usual procedures for crystalllzatmn of water soluble proteins, such as increasing ionic strength by vapor dfffusmn, were not apl'hcable for this enzyme C ~ s t a l s of the resting oxidized enzyme belong to a space group of P62 or P64 wath cell dlmensmns, a = b = 208 7 A. and c = 282 3 A The Patterson function shows that the crystal exhibited a non-crystallographic two-fold axis parallel to he c-ards in the asymmetric umt

I. Introduction Cytochrome c oxadase is a key enzyme xn bIoenergettcs in that it catalyzes the reduction of m(lecular oxygen to water and pumps protons acloss the tuner mitochondrlal membrane [1]. The en, ymes of a wide range of aerobic orgamsms ha,~e been isolated and show many structural slrmlar ties [2]. The bovine enzyme has been the most th(roughly studied. It has been reported that the bo'nne enzyme contains 12-15 different polypel)tlde subunits [3,4]. Organ specificity for the su[,unlt composition also has been suggested [5]. Re,zently Einarsdottlr and Caughey found that the enTyme contains Fe, Cu, Zn and Mg In the atom ratios of 2 : 2.5 • 1 : 1 [6,7]. Metal analyses of crystalhne enzyme confirmed thexr results [8] while

Buse and co-workers claimed a 2 : 3 atom ratio for Fe : Cu [9]. Identification of the subunits in whach the metals are located is incomplete, but some evidence points to the two hemes and two of the coppers as being associated with subumts I and II

[1]. The three-dimensional structure at high resolunon by X-ray crystallography is most desirable information for elucidation of the mechamsm of this enzyme. However, integral membrane proterns have proven very difficult to purify and crystallize [10]. Therefore, the crystalhzation of this enzyme has been one of the most important and challenging subjects m the study of the enzyme. Recently we succeeded in making crystals of bovine heart enzyme which diffract X-rays at a resolution of 8 ,~ [8]. Reports on the crystalhza-

0 0 2 J - 0 2 4 8 / 9 1 / $ 0 3 50 '~ 1991 - Elsevier Science Pubhshers B V (North-Holland)

248

S Yo;lukawa et al /' Crv;talhzat*on o f b e e / h e a r t c~tochrome ~ ovtda~e

non of cytochrome c ox~dase prior to our previous report turned out to be lrreproduclble [11,12] Only two other m e m b r a n e proteins from eukaryotlc organisms, light-harvesting chlorophyll a / b protem complex and prostaglandIn H synthase, have yielded crystals which show X-ray diffraction patterns [13,14]. Here we report the recent developments of the method of crystalhzatlon and the crystallographic analyses for bovine heart c)tochrome c oxadase in the fully-oxidized restmg state.

2. Materials and methods

C~tochrome c oxadase was isolated from bowne heart muscle by the method of Yoshlkawa et al [15], using Bnj-35 (Pierce Chemical high purity detergent) as the non-ionic detergent unless otherw~se stated. Small monodlspersed, non-~onlc detergents, e.g., several alkyl oxyethylene species (Nikkol), octyl glucoside, lauryl maltoside (Sigma) and LDAO, were used for some preparations instead of Brlj-35 (polydlspersed non-ionic detergent) The final product in the oxidized (resting) state, freely soluble in neutral buffer without any added detergent, was further purified by microcrystalhzation with an ultrafiltratIon apparatus as described m a previous paper [8] The crystalhne enzyme preparation as ~solated was used in this study. Enzyme concentration is expressed in terms of heme A measured by optical spectra. For making hexagonal blpyramldal crystals suitable for X-ray diffraction, sohd Brij-35 was carefully added stepwlse to the enzyme solution (typically, 1.0 ml) 0.6raM 0.8mM in heme A, until a shght turbidity due to crystalhzation appeared After addmon of a nunlmal volume of the enzyme solution without the detergent to dissolve the turbidity, the enzyme-detergent mixture was placed in 8-10 small quartz test tubes (2 × 20 mm) Then, fine adjustment for the detergent concentration was made for each test tube by adding a measured volume of the enzyme solution either with or without added detergent A series of enzyme detergent mixtures containing various concentrations of the detergent at a constant protein concentration, thus prepared, was allowed to stand

for several days at 4°C. This procedure was required for each batch of enzyme preparation since the best detergent concentration vaned from preparation to preparation.

3. Results and discussion

The e n z y m e crystallized instantaneously without any coexisting amorphous materml when crystallization conditions were attained. The crystal system depends on buffer concentration and buffer specms, as shown m table 1 Hexagonal blpyramldal crystals (fig 1) were obtained in the lower concentration range of each buffer whereas tetragonal plate crystals (fig. 2) appeared in the higher concentration range. The concentration ranges depended on buffer species. In Tns-HCI buffer, tetragonal plate crystals were not obtained In each case crystals appeared more readily at htgher protein concentrauon, lower ionic strength (wittun each concentration range as given in table 1), higher detergent concentration, and lower temperature As described in the previous paper [8], the enzyme was crystalhzed from the protein solution without added detergent by concentrating the enzyme solution w~th an ultraflltratlon apparatus. The best interpretation for the result is that the residual detergent in the enzyme soluUon was concentrated with the ultraflltration apparatus Nonpenetration of Bru-35 through the ultraflltrauon membrane was detected. As we reported previously [8], in order to obtain large hexagonal blpyramldal crystals, the enzyme solution was concentrated to near the cr~stalhzatlon point with an ultraflltratmn apparatus and allowed to stand for several days at 4°C However, the deternunatlon of crystalhzaUon point turned out not to be reproducible, and so it was very difficult to find out the best point to stop concentrating the enzyme solution On the other hand, concentrating the enzyme solut,on slowly without stirring, in order to approach the best concentration without determining it, was not effective for getting large cr~ystals. Perhaps concentratlons of the detergent and the protein were increased fairly rapidly near the surface of the ultraflltrat~on membrane to mduce formation of

S Yoshlkawa et a l /

CrFstalhzatlon of beef heart cvtochrome c oxzdase

249

Fig 1 Hexagonal blpyrarmdal crystals of cytochrome c oxldase The mother liquor contained 5 m M Hepes buffer pH 7 4 The biggest crystal in the photograph is about 0 3 m m m the largest dimension

too m a n y mlcrocrystals. Trials for c r y s t a l h z a t l o n wit a decreasing buffer c o n c e n t r a t i o n b y v a p o r dlffusion or m i c r o d i a l y s l s were n o t successful be-

cause a c o n c o m i t a n t decrease in the p r o t e i n and the d e t e r g e n t c o n c e n t r a t i o n s was n o t avoidable. I n c r e a s i n g the d e t e r g e n t c o n c e n t r a t i o n b y a d d i t i o n

t:qg ~_Tetragonal plate crystals of cytochrome c oxldase in 30mM Na-phosphate buffer pH 7 4 The size of the plate is about 0 03 m m m the largest d~mensmn

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S Yo~h*hawa et a l /

Cr~ ~talhzatton of beef heart Lvtochrome ~ owda~e

Table 1 Effects of buffer specms and concentration on the crystal system Crystal system

K-phosphate Na-phosphate Tns-HC1 Hepes Mops

Hexagonal

Tetragonal

lmM-3mM lmM-5mM lmM-5mM lmM-10mM lmM-10mM

4 m M - 10mM 6mM-50mM ND ND

pH 7 4, protein concentration 0 5 m M - 1 l m M

of the detergent (in solid state or dissolved in the enzyme solutton) seems the easiest method for changang only one parameter for crystalhzation while keeping the other constant Therefore, the method described in sectton 2 is reqmred for obtaining large crystals ( > 0 6 mm) reproduclbly from any batch of the preparation A typical result is given m table 2. The crystal size is very sensmve to detergent concentration. In this case, the detergent concentration in the enzyme detergent mixture without fine adjustment, i.e., solution A alone without additions, yielded one of the best results

Table 2 Fme adjustment of the detergent concentratton Enzyme solutions (Bnj-35) "* (>l) A B C

Bnj-35 (final

Size of cr'~stals

(1 9 5 , % l

(000%)

concentrat~on )

(mm)

100 100 100 100 100 100 100 100 100 100

5 4 3 2

(5 87%)

2 4 6 8 10

1 86 1 88 1 89 1 91 1 95 2 03 2 10 2 17 2 24 2 31

03 05 0 5 04 4}3 03 < I) 05 < 0 05

~ Enzyme concentration 0 713mM in heme A

Buffer species also affected the quality and the size of hexagonal blpyramldal crystals Hepes buffer pH 7.4, 5mM, was the most statable buffer for the crystalhzatton so far examined. The present method for crystalhzatton has improved the s~ze and shape of the hexagonal bi-

Fig 3 Tetragonal plate crystals of cytochrome c oxldase containing lauryl octaoxyethylene instead of Bru-35 m 30mM Na-phosphate buffer 7 4 The size of the plate is about 0 5 mm m the largest & m e n s k m

S Yoshtkawa et al / Co'stalhzatlon of beef heart qvtochrome c o:~tdase

p~ ramadal crystals very much (compare fig. 1 with fig. 1 in ref. [8]). More than 80% of the ideal reJlectlon numbers in the range of 7 A resultlon were obtained at the acceptable cntera, F 2 > 30(F2), at the Photon Factory, National Laborat o ' y of High Energy Physics, Tsukuba, Japan. The sp ace groups and cell dimension are P62 and P64 an~ a = b = 2 0 8 . 7 A, c = 2 8 2 . 3 A, respectively. T i e Patterson function showed clearly a noncu'stallographxc two-fold symmetric axis parallel to the c-axis in the asymmetric unit, suggesting thlLt the enzyme is in a dlmenc state. The crystallo~raphlc studies will be pubhshed in detail elsewhere. However, the resolution of the X-ray dllfraction pattern was still fairly low (7 A). One of the possible factors lowering the quality of the cr 3 stal may be the polydispersed detergent, Bnj-35, tlgatly bound to the enzyme molecule The cont e r t of the detergent is about 25% of the total m(lecular weight [8]. Therefore, we tried to replace Bnj-35 with the monodlspersed small detergei Lts often used for various membrane protein cr 3 stalhzations as given in Materals and Methods. However, no detergent examined, except for do, lecyl octaoxyethylene (Nikko Chermcal), provld ed a good preparation which was stable against de~Laturatlon. Not even mlcrocrystals were obtained from these preparations. However, tetragonal plate crystals were obtained from the enzyme preparations contaimng dodecyl octaoxyethylene. Th,; large crystals, as shown in fig. 3, diffracted X-rays at fairly low resolution (15 ,~). The low res,)lutlon seems mainly due to the unstable propertJes of the enzyme preparation. Therefore, imprevement of the isolation method in the presence of detergent would improve the quality of the cry,;tal. Hexagonal crystals were not obtained from this enzyme preparation. The possibdlty that heterol,ene~ty in peptlde subunlt structure affects the res~,lutlon of the diffraction pattern cannot be

251

excluded. C o m m o n methods for crystalhzauon of water soluble proteins, such as vapor diffusion and microdlalysls using a m m o m u m sulfate or polyethylene glycol as preclpxtant, have been unsuccessful.

Acknowledgements This work was supported in part by a Grant-inAid for Scientific Research 01580265 from the Ministry of Education, Science and Culture of Japan (S.Y.) and United States Pubhc Health Service G r a n t HL-15980 (W.S.C)

References [1] G Palmer, Pure Appl Chem 59 (1987) 749 [2] M Wlkstrom, K Krab and M Saraste, Cytochrome Oxldase - A Synthesis (Academic Press, New York, 1981) [3] N W Downer, N C Robinson and R A Capaldl, Blochermstry 15 (1976) 2930 [4] B Kadenbach, J Jarausch, R Hartman and P Merle, Anal Bxochem 129 (1983) 517 [5] R A Capaldl, Trends Blochem Scl 13 (1988) 144 [6] O Emarsdottw and W S Caughey, Blochem Blophys Res Commun 124 (1984) 836 [7] O Emarsdottlr and W S Caughey, Blochem Blophys Res Commun 129 (1985) 840 [8] S Yoshakawa, T Tera, Y Takahashl, T Tsuklhara and W S Caughey, Proc Natl. Acad. Scl USA 85 (1988) 1354 [9] G C M Steffens, R Blewald and G Buse, European J Blochem 164 (1987) 295 [10] R M Garavato, Z. Markovac-Housley and J A. Jenkins, J Crystal Growth 76 (1986) 701 [11] T Yonetam, J Biol Chem 236 (1961) 1680 [12] T Ozawa, M Tanaka and Y Wakabayashl, Proc. Natl. Acad. Scl USA 79 (1982) 7175 [13] W Kuhlbrandt, J Mol Blol 194 (1987)757 [14] D Picot and R M Garavato, m Cytochrome P-450 Blochen~stry and Biophysics, Ed I Schuster (Francis and Taylor, London, 1988) pp 29-36 [15] S Yoshlkawa, M G Choc, M C O'Toole and W S Caughey, J Blol Chem 252 (1977) 5498