Crystallographic study of rabbit liver cytochrome b5

421

B I O C H I M I C A ET B I O P H Y S I C A ACTA

PRELIMINARY

NOTES

BBA 3 I O 7 o

Crystallographic study of rabbit liver cytochrome

b 5

Cytochrome b5 is a promising protein for high-resolution X-ray crystallographic study. As MATHEWS AND STRITT.~IATTERa noted in their description of crystals of calf liver cytochrome b 5, it, like myoglobin, contains a noncovalently bound heine group, whose iron atom is probably coordinated by two histidine side chains. However, in contrast to myoglobin, it does not bind oxygen. Hence a comparison of its tertiary structure with those of myoglobin and of cytochrome c might allow one to determine the mechanism whereby it performs its function of transferring electrons. TSUGITA and co-workers2, 3 compared the primary structures of rabbit, human and chicken cytochromes b5 with that of calf4; they found several common and variable regions in the molecule. Hopefully the detailed effects of such differences will be seen in a comparison of the tertiary structures of bovine cytochrome bs, now being investigated by MATHEWS AND .R,TRITTMATTER 1, with that of rabbit cvtochrome b5 as described here. Crystals of rabbit liver eytoehrome b~ * were grown by dialysis against 8oC),o saturated (N1-14)2S0a buffered with phosphate from pH 5.5 to 7.0. These crystals were stable when transferred to 3.8 M phosphate; however we were unable to grow large single crystals directly from phosphate. The crystals are lath shaped, with maximum overall dimensions o.z mm (a axis) o.2 mm (b axis) by I.O nun (c axis). It is interesting to note in Table I that even though both the native enzvmes from rabbit and calf crystallize in space group P,~12t2~, there is no discernible relationship between their ~~

, :

.

', ."

¢

I.I

.~..:

.;,

I

¢

g

Fig. r. A n t 8 ° p r e c e s s i o n p h o t o g r a p h

of t h e hko z o n e of a r a b b i t l i v e r c y t o c h r o m e b s c r y s t a l . Biochim. Biophys. .4cta, 2oo (i~7o) 4-,1 -422

422

PRELIMINARY NOTES

TABLI- I CYTOCHROME b 5 SPACE GROUPS AND UNIT CELL DIMENSIONS

Axis Space group . a b c Volume per asymetric unit

Rabbit Pzl212!

Calf (r@ l) P212121

II1.X :~, 36.3 -~ 5"1.4 .~.

64.55 :k 46.Ol .-~ 29.89 :k

5i 840 .~3

22 x9o :ks

respective packing arrangements. In fact the rabbit enzyme has two molecules per unit cell while calf liver cytochrome br, has only one. Strong reflections are obserw~d to 2.5 A ° resolution, see Fig. i. The crystals are reasonably stable to X-radiation. For instance about 4 ° h exposure is required for a 18 ° precession p h o t o g r a p h ; the X - r a y diffraction pattern shows signs of deterioration only after 12o h of crystals X-radiation. This research has been supported by The Center for Advanced Studies, University of Virginia, by N.I.H. Grant 5 ROI GM 15786-o2, and grants of the .]apanese Ministry of Education. The assistance of I)orothae Dangelat is gratefully acknowledged.

Department of Biology, University of Virginia ('harlottesville, Va. 22903 (U.S.A.)

R. H. KRETSINGER

Department of Biochemistry and Laboratory of Molecular Genetics, Medical School, Osaka University, Kitaku, Osaka (Japan)

BUNJI HAGIHARA AKIRA TSU(;ITA

I F..'~. ~IATHEWS AND P..~TRITTblATTF.R, J. Mol. Biol., 47 (19(K)) 2952 .'\. TSUGITA, M. KOBAYASItI, "['. KAJIHARA AND |~J. HAGIHARA, J . Biochem. Tokyo, 64 (I968) 727 . 3 A. TSUGITA, M. KOBAYAStII, .~,. TANI, .~. KYO, M. A. RAStIID, Y. YOSHIDA, r . KAJIHARA AND ]{. HAGIIIARA, in p r e p a r a t i o n . .~ T. KAJIHARA AND }aI. HAGIIIARA, .]. Biochem. Tokyo, 63 (I968) 4535 J- OZOLS AND P. STRITT.~IA'I'TER, ] . Biol. Chem., 24t (I966 t 4793.

Received November 5th, I96q Biochtm. Btophys. Acta, 200 ( 197 o) .ta 1-422