A hydrogen-deuterium exchange study of membranous cytochrome oxidase

Biochimica et Biophysica Acta, 303 (I973) 2 3 7 - 2 4 I © E l s e v i e r Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s

BBA 36369 A H Y D R O G E N - D E U T E R I U M E X C H A N G E STUDY OF MEMBRANOUS CYTOCHROME O X I D A S E R O D E b t I C K A. C A P A L D I

University of Wisconsin, Institute for En~yrne Research, x7ro University Avenue, Madison, Wise. 537o6 (U.S.A.) ( Receiv ed N o v e m b e r 6th, z972)

SUMMARY

The rate of exchange of peptide hydrogens of membranous cytochrome oxidase for solvent deuterium has been followed by infrared spectroscopy. The infrared spectrum indicates that the enzyme contains g helix and possibly fl conformation. Tile exchange data show that a m a x i m u m of 59% of the polypeptide chains are in these conformations. Also, the data indicate that at least 6o% of the cytochrome oxidase is exposed to the aqueous milieu which surrounds the membrane.

INTRODUCTION

Optical rotatory dispersion, circular dichroism, infrared spectroscopy and hydrogen exchange studies have all contributed to our understanding of the conformation of proteins in aqueous solution. Most of these techniques have been used to study the structure of membrane proteins but with only limited success 1 3. The data from optical techniques such as circular dichroism and optical rotatory dispersion are difficult to interpret because of artifacts of light scattering 2, while infrared spectra give at best only qualitative information about the presence or absence of a helical and fl conformation in proteins 3. At present there is little hope of obtaining detailed X - r a y data on membrane proteins and our knowledge of the structure of these proteins requires collation of the information provided by a wide variety of techniques. This communication describes the application of a hydrogen-deuterium exchange technique to the study of membrane systems. Infrared spectroscopy has been used to follow the rate of exchange of peptide hydrogens of membranous cytochrome oxidase with solvent deuterium. Results indicate the feasibility of using this technique for membranes. Further the data add to our understanding of the structure of the cytochrome oxidase complex in the membrane. MATERIALS AND METHOD

Membranous cytochrome oxidase was prepared by a modification of the method of Sun et al. 4 which has been described recently by Vanderkooi et al. 5. This mem-

238

R.A. CAPALD1

b r a n o u s p r e p a r a t i o n , containing between 27 °/o a n d 3 3 /o/o lipid b v weight, was washed three times with IO mM Tris-HC1, p H 7 . 4 , to remove most of the T r i t o n X - I o o , and finally suspended to 2o mg p r o t e i n / m l in the same buffer. 2-ml aliquots (4 o rag) of the oxidase were p i p e t t e d into homogenizing vessels and these samples were freezedried d i r e c t l y in the vessels. Lyophilized samples were stored in a v a c u u m dessicator prior to use. E l e c t r o n microscopy confirmed t h a t the p r e p a r a t i o n regained its vesicular, m e m b r a n o u s c h a r a c t e r when suspended in 2HeO after lyophilization. Also representative, lyophilized c y t o c h r o m e oxidase samples were e x a m i n e d s p e c t r o p h o t o m e t r i c a l l y after suspension in 2H20 to ensure t h a t the p r o t e i n h a d not been d e n a t u r e d b y the m a n i p u l a t i o n s required to o b t a i n infrared spectra. The oxidized, reduced, a n d reduced v e r s u s oxidized difference s p e c t r a h a d the characteristics of the n a t i v e enzyme, i.e. a p e a k at 421 n m in the oxidized state which shifted to 442 n m in the r e d u c e d state, a n d a p e a k at 6o5 n m which was g e n e r a t e d in the r e d u c e d vers~ts oxidized difference s p e c t r u m 6. S p e c t r a were o b t a i n e d using a P e r k i n - E l m e r Model 521 double b e a m g r a t i n g infrared s p e c t r o p h o t o m e t e r , o p e r a t e d in the n o r m a l mode. C a F 2 cells of o.o 5 nun p a t h length were e m p l o y e d . Samples were injected into the cells with a I - m l syringe, care being t a k e n to avoid air b u b b l e s in the t h i n film of sample. Vesicles were r e c o n s t i t u t e d a n d exchange was i n i t i a t e d b y homogenizing indiv i d u a l samples of the freeze-dried c y t o c h r o m e oxidase (4o mg) in i ml of 2H20. The buffer was freeze-dried into the sample ; t h u s the conditions of exchange were IO mM Tris 2HC1 (p2H 7.8) (p~H - - p H + o.4o)L The s t a r t i n g t i m e for each e x p e r i m e n t was t a k e n as the i n s t a n t at which the ~H20 was added. H o m o g e n i z a t i o n t o o k between z a n d 2 min. A f t e r s a t i s f a c t o r y suspension of the sample, an aliquot was i m m e d i a t e l y t r a n s f e r r e d to the C a F 2 cells and a m e a s u r e m e n t was t a k e n . The rate of h y d r o g e n exchange was m e a s u r e d b y following the decrease in the absorbance of the a m i d e I I b a n d (154 ° cm -1) as a function of time, using the a m i d e I b a n d as a measure of the c o n c e n t r a t i o n of sample 8. The amide I I b a n d was scanned first to minimize the h e a t i n g effect of the infrared beam. The s p e c t r o m e t e r was set at a w a v e l e n g t h of I555 cm -1 a n d a scan speed of i cm -1-S -1 before the sample was inserted in the infrared beam. A scan was m a d e i m m e d i a t e l y after the sample was placed in the beam. The times i n d i c a t e d in Figs I a n d 2 are the times at which the scan passed over the p e a k at I54O cm ~. The sample was t h u s in the infrared b e a m only 15 s before a m e a s u r e m e n t was o b t a i n e d a n d in this short p e r i o d of t i m e the h e a t i n g effect of the b e a m is negligible. Thus the t e m p e r a t u r e of the exchange e x p e r i m e n t was the 2o ± I °C at which the stock solution was m a i n t a i n e d . Once t h e absorbance of t h e a m i d e I I b a n d h a d been measured, a scan from 175o to 145o cm 1 was o b t a i n e d from which the dimensions of the amide I b a n d were calculated. B l a n k m e a s u r e m e n t s were m a d e with each d e t e r m i n a t i o n . Several d e t e r m i n a t i o n s were m a d e on s e p a r a t e aliquots over a 72 h period. The absorbance of the a m i d e I I a n d a m i d e I b a n d s of u n e x c h a n g e d m e m b r a n o u s c y t o c h r o m e oxidase were o b t a i n e d from s p e c t r a of a p r e p a r a t i o n in IO mM Tris HC1 (pH 7.4) which h a d been lyophilized onto tile windows of the C a F 2 cells as a thin film. The absorbance of the a m i d e I I and a m i d e I b a n d s of c o m p l e t e l y d e u t e r a t e d samples were o b t a i n e d from s p e c t r a of samples which h a d been h e a t e d for I min at IOO °C, in a solution of 2H20 which was 3 % in sodium dodecyl sulphate.

MEMBRANOUS CYTOCHROME OXIDASE

239

Tile percent of peptide hydrogens unexchanged, Ht, at time t~ was obtained from tile following relationship. ta

A 1540tI

A 1540

"~ 1654tl

A1045 t a

Z~ 154010

~ 1540 t~

,4 1050t0

~ 1045 t a

St =

X ioo

where A~a4oti/Axs54tl is the ratio of the absorbance of the amide I I to that of amide I band at time q. A~5aot~/A1¢45 t~ is the ratio of the absorbance of the amide I I to t h a t of amide I band after exchange of all the peptide hydrogens, and Az54oto/Als5otois tile ratio of the absorbance of the amide II band to t h a t of amide I band before exchange has commenced. Ala40t~/A1645t~~ w a s measured to be o.I8 and A154oto/A1654towas obtained as o.65. Thus I N 1540t

1/"~ 1,;50t 1]

O. I 8

Ht-

X too o.47

RESULTS AND DISCUSSION

Infrared spectra of membranous cytochrome oxidase in 2 H 2 0 ( I o mM Tris 2HC1, p~H 7.8) at different stages of exchange are shown in Fig. I. The amide I band centered close to z65o cm 1 corresponds to tile C = O stretching frequency of the peptide bond". There is some evidence to indicate t h a t this band is sensitive to the conformation of proteins~°, 11. In 2H20 solution, the peak is centered around z654 cm -1 for proteins in the a-helical conformation, x645 cm -x for proteins in a r a n d o m coil, and around z633 cm ~ for proteins in the fl conformation. In membranous cytochrome oxidase the amide I band is centered around z654 c m - L indicating an a-helical arrangement of a significant portion of the protein. There is also a shoulder around 90-; .e

i i

(n z E i-~

i

I

I

{

I

:....

L

WAVELENGTH c m-1

Fig. i. I n f r a r e d s p e c t r a o f m e m b r a n o u s c y t o c h r o m e o x i d a s e a f t e r d i f f e r e n t t i m e s o f e x c h a n g e . T h e s p e c t r a a r e as f o l l o w s : - - , 3 m i n ; . . . . . . , 6o r a i n ; - - - - - , complete exchange after heating t h e s a m p l e w i t h 3 % s o d i u m d o d e c y l s u l p h a t e f o r I m i n . T h e a m i d e I b a n d a r o u n d 165o c m -1 w a s s c a n n e d w i t h t h e c h a r t m o v i n g I c m f o r e v e r y 4 ° c m -4 o f t h e s p e c t r u m . B e l o w 16oo c m -1 t h e c h a r t speed was c h a n g e d to record ioo c m - I per c m of chart.

240

R . A . CAPALDI

1625 cm -1 which may represent a small amount of fl structure in the enzymic complex. Upon solubilization in 3% sodium dodecyl sulphate which unfolds the native conformation of the enzyme, the amide I band shifts to a lower frequency i.e. from 1654 cm -~ to 1645 cm 1. This shift is typical of enzymes with a high proportion of a helix such as myoglobin and hemoglobin but is not observed in proteins with a low proportion of ¢ helix such as a caesin and ribonuclease (Capaldi, R. A., unpublished observations). Tile peak at 158o cm ] corresponds to the C = O stretching frequency of ionized carboxyls x~ while tile band centered around 154o cm -1, tile amide II band, arises from a coupled C N stretching and N H deformation frequencyl2,14. As tile amide hydrogens from the protein backbone are exchanged for deuterium, this band disappears and a new band forms around 145o cm 1. The 145o cm 1 band arises both from deuterated peptide groups and 2HHO formed in the exchange reaction x4. The extent of exchange of the peptide hydrogens after various times of incubation is shown in Fig. 2.41°/o of all peptide hydrogens exchanged before a measurement was taken (at

!8o

--0.65

60--0.41

x~ 40--~o ~°o

n ~

~

~

I

~ 1

_ _

20-

I 1

I

2

]

I

3

4

TIME

IHOIJRS)

0.18

5 1272

Fig. 2. The rate of exchange of the peptide hydrogens of m e m b r a n o u s c y t o c h r o m e oxidase. The percent of unexchanged hydrogens is plotted at times up to 7 2 h. The continuous line represents the best curve t h a t can be d r a w n by eye t h r o u g h the d a t a points. Different s y m b o l s represent different e x p e r i m e n t s on separately p r e p a r e d samples of enzyme.

9° s). Another 19% exchanged during the following 72 h of incubation, most being exchanged within 3o rain of suspension. 4o% of all the peptide hydrogens did not exchange during the course of the experiment. Exchange studies of soluble proteins whose three dimensional structures have been determined by X-ray methods, have shown that only hydrogens which are exposed to solvent and are not hydrogen bonded, exchange very rapidly. Hydrogens which are involved in hydrogen bonds such as those that stabilize ~ helices and fl pleated sheet and hydrogens which are deeply buried in a hydrophobic environment, exchange much more slowly15 17. Thus for membranous cytochrome oxidase, 4I°,~ of the peptide hydrogens must be accessible to solvent and not involved in hydrogen bonds. Another 19% of the peptide hydrogens must be accessible to solvent although retarded from exchanging, most likely because of their involvement in hydrogen bonds. The remaining 4 ° % of the peptide hydrogens must be hydrogen bonded and/or

MEMBRANOUS CYTOCHROME OXIDASE

24I

deeply buried in the hydrophobic interior of the membrane. The fact that 41% of all the peptide hydrogens exchange very rapidly, puts an upper limit of 59% on the amount of a helix plus fl structure in the enzymic complex. Also, the fact that 6o% of all the peptide hydrogens exchange with deuterium indicates that at least this amount of the enzymic complex is exposed to the aqueous milieu surrounding the membrane. This percentage of exposed protein is more than sufficient to accommodate all the charged groups and indeed all the polar groups of the enzyme which would prefer to be in an aqueous rather than a hydrophobie environment (37% of the amino acid residues~8). Thus the notion that intrinsic membrane proteins, including cytochrome oxidase, are amphipathiclg, 2° is not prejudiced by the hydrogen exchange data. Also, the extent to which the enzyme complex is exposed to water provides considerable surface for interaction with cytochrome c, an extrinsic protein19, 2° which is associated with the surface of the membrane by predominantly electrostatic interactions. ACKNOWLEDGEMENTS

I am grateful to Dr David E. Green for his encouragement and for helpful discussions during the course of this work. Also, I am grateful to the Wellcome Trustees for the award of a Wellcome Research Travel Grant. This work was funded by grant GM-I2847 from the National Institute of General Medical Science (U.S.P.H.S.). R E FERENCES I 2 3 4 5 6 7 8 9 to II 12 13 14 15 16 17 18 19 2o

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