Structure of beef liver catalase

.J. Mol. Rid.

(1981) 152. 46.5-499

Structure Iv~.+~~HvK R.

of Beef Liver Catalase

N. MVRTHY, THOMAS ,I. REID KOBUO TAKAKA$ AKD MICHAEL

III?, 6.

ANDREW

SICIGNANO~

kHShfANN\\

Departments of Biological Sciences ad Hiorhrmistry Purdlre tTniversity, M.‘pst IAfayette, Ind. 47907, Tj’.I(‘.A. (Rrcriwd

.5 May

19X1)

The t,hree-dimensional struct’ure of beef liver catalase has been determined to 2.5 AA resolution I)y a combination of isomorphous and molecular replacement techniques.

Heavy-atom

positions

were found using vector search and difference

Fourier

methods. Thr tetrameric catalase molecule has 222 symmetry with one of its dyads coincident with a crystallographic P-fold axis. The known polypept,ide sequence has bratsn unambiguously fitted to the electron density map. The heme is well buried in a hydrophobic pocket. 20 A below t,he surface of the molecule, and accessible through a hydrophobic channel. Residues that line the heme pocket, belong to two different subunits. Tyr357 is the proximal heme ligand and the catalytically important residues on t)he distal side are residues His74 and ilsn147. The tertiary structure consists of four domains: an extended non-globular amino-terminal arm, which stabilizes t.he quaternary strurture: an anti-parallel, eight-stranded o-barrel providing the residues on the distal side of the heme: a rather random “wrapping domain“ around thca subunit exterior including the proximal heme ligand ; and a tinal I-hrlic,al structure resembling the E, F, (: and H helices of the globins.

1. Introduction (‘at,alasca (EC 1.11.1.6, hydrogen peroxide : hydrogen peroxide oxidoreductase) occurs in almost all aerobically respiring organisms and serves to protjrct cells from the toxic effects of H,Oz by catalyzing the reaction: %H,O,

-+ 2H,O + 0,.

(1)

Ohligatt> anaerobes cannot tolerate 0, in part because they lack this enzyme. The “catalat,ic” activity (reaction 1) distinguishes catalase from other peroxidases ((‘hancv. 1948) except for chloroperoxidase, which ca,talyzes a wider variety of substratjes (Hewson Br Hager, 1979). Catalase can also oxidize a variet,y of c~ompounds in the presence of H,O, (Keilin & Hartrer, 1955). This “peroxidatic” t Present address: Uniformed Services. I’niversity of the Health Sciences. Bethesda. Md. C’.S.A. : Present address. Charter International Oil Co.. P.O. Box 5008. Houston. Tex. 77012. 1W.A. 5 Present address: Institute for Protein Kesparch, Osaka I-niversity. R311. Ysmada-kami. Suit.;l. Osaka. Japan. 11To whom reprint requests should be addressed at the Ijepartment of Biological Sciences. Purdue I’nivrrsity, a’est Lafayette. Ind. 47907. I..S..I\. M)%2-~2XWX 1t3004H5-35 $02.00/0

+ti5

‘CT 1981 Academic Press Inc. (London)

Ltdi.

M. It. S. MlJRTHY

466

activity

(reaction

ET

AL.

2) : HzOz+XH,

+ .I,+:!H,O.

(2)

is responsible for ethanol oxidation in liver. provided t,he H,O, concentjration is sufficiently high (Chance et al., 1974). (‘atalase contains four identical subunits (Tanford & Lovrien, 1962: Sund rt (II., 1967) each equipped with a high-spin Fr(lII)protoporphyrin IX (Theorell & Agner, 1943: Torii it al., 1970: Stern. 1936). However. approximat’ely one of the four subunitas contains an inactive high-spin Fe(lII)-biliverdin complex (Sumner & Douncr. 1937: Lemberg &, Legge. 1943: (‘hang & Schroeder, 1972). Low-angle Xray scattering data showed beef liver catalasr to be an ellipsoidal molecule with a 40 a radius of gyration (Malmon. 1957). One subunit of vatalase has 506 amino acid residues with an additional 10 to 15 residues at t’hr (‘-terminal of a small fract,ion of the subunits (W. A. Schroeder, personal communication). The f\‘-terminus is blocked by an as yet unidentified group (Schroeder Pt al.. 1969). The 16 cysteine residues in the tet,rameric mammalian catalasr molecule do not form disulfide bridges (Miirikofer-Zwez et a/.. 1969: Pihl PI cl/.. 1961). (‘rystals of catalase were observed as early as 1937 by Sumner 8 Douncr (1937). Reports of crystalline catalase from lamb (Dounc~e. 1942), horse (Bonnichsrn, 1947a) and human (Bonnichsen, 19476) liver. from beef (Laskowski &. Sumner. 1941). horse (Bonnichsen, 1947a) and human (Bonniuhsen, 1947a,: Herbert, B Pinsent, 194%) erythrocytes. from spinach leaf (Galston et al., 1951) and from bacteria (Herbert & Pinsent, 19486) followed. Electron microscopy (Hall, 1950 : Valentine,.1964: Kiseler d al.. 1967) and elect’ron diffraction (Matricardi et al.. 1972 : Taylor Rr Glaeser, 1974: Unwin, 1975) investigations of complexes wit,h a habit similar to that obtained by Sumner & Dounce (1937) revealed an orthorhombiv lattice with space group P2,2,2,. Human erythrocyte and beef liver catalas? can also form cylindrical t*ubules (Kiselev d ~1.. 1967). The first cat,alase crystals to be studied by single-crystal X-ray diffraction were a disordered ort,horhombic form of horse erythroryte catalase (Glauser & Rossmann. 19fjfi). Subsequently, t,wo crystal forms of href liver catalase suit,able for highresolution structure determination were reported : the first, was a trigonal form of space group P3121 or P3,21 with six molecules in the unit’ cell (Labaw. 1967: Rossmann Cy:Labaw. 1967; Longley, 1967: Gurskaya r! al.. 1971) and the other was one molecule in the an orthorhombic form of space group P2 1212,, containing & Rich, 1973). Rurkey & asymmetric* unit (Gurskaya et al.. 1971 : McPherson catalase crystals are McPherson (1977) showed that beef and deer liver isomorphous. Eventoff 8r Gurskaya (1975) were unable to distinguish between t’hc point groups D, and C, for the molecular symmetry in a rotation fun&ion study with 10 /! resolution d&a of beef liver caatjalase. The possibility of a i-fold discounted as catalase dissociates into dimers and symmetry was, however. monomers (Tanford & Lovrien, 1962). This was [*onfirmed in other crystal forms by t,hr incorporation of a crystallographic P-fold axis in t,he molecule of beef liver catalase (Eventoff et al.. 1976). Pmicilliurn citalr catalase (Vainshtein et al., 1979) and Micrococcus lutrws catalase (Marie et al., 1979). The structure determinat’ion of the beef liver catalase crystals report,ed by Event,off rt al. (1976) is reported here.

STRVC’TVRE

OF BEEF

LIVER

-Ifi7

(‘.4TALASE

2. Experimental Beef liver caatalase was prepared as descbribed previously (Eventoff ut al.. 1976: Reid it al.. 1981). The heavy-atom compounds used for isomorphous replarement and their soaking conditions are listed in Table 1. X-ray diffraction data were recorded on Ilford CTX-ray films for the native, sodium mersalyl and K,PtCI, derivatives and on Kodak no-screen X-ray films for the Hg(‘l, and dimercury acetat,e rrystals. Data w-ere collected with an Enraf: Sonius rotation camera using CuKa radiation from an Elliott GXR rotating anode X-ra> generator operating with a 100 pm fhcal cup at 35 kY and 20 mA. The radiation was filt,rrrtl 1)~ a 157 pm thick nickel foil attached to a 0.25 mm caollimat,or. The crystal-to-film distanccb was 75 mm. Two dat)a collection schemes were employed. In the first, method, used for thr native. mrrsalyl and K,PtCI, derivatives, the n*-axis was placed parallel to the spindle axis of t,ht, camera : in the second method. used for HgCI, and Hg,Art derivatives, the c*-axis was taken as the osc*illation axis. This required data to be collected over a 90” angle for the former case and only 30” for the latter. Cryst’al morphology required the development of a bridle to handle the c* mount. An oscillation angle of 1.5” provided a good compromise between thts num her of’ whole, non-overlapped reflections and overlapped reflecations on any given film. ‘I’AHLE

1

Heavy-atom soakiny conditiot~s Abbreviation

(‘oncentration (mM)

Soaking timp (days)

The oscillation angle between successive films overlapped 03” in order to provide a sufficient number of common reflections for film-film scaling. Each exposure for the native crystals la&d (i h and for derivative crystals 83 h. Four separate exposures were collect,ed from earh cnryst,al. The films were digitized on an Optronics film scanner at intervals of 100 pm in the range of 0 to 2 ON. units divided into 2FiB gray levels. Crystal orientations were refined by the c*onvolution met,hod and intensities were estimated by profile fitt’ing (Rossmann, 1979). The metShod of Hamilton rt nl. (1965) was used for initial scaling of films in a film pack and subsequent scaling relating the film packs to each other. Anisotropic scaling factors wer(a c*alculat~ed for each film using only those intensities greater than 2 standard errors. lntrnsities less than 1 standard deviat,ion were rejected from the final data set. The other rejection criteria and detailed techniques have been described elsewhere (Rossmann a/ nl.. 1979). The csrystal orientations and the unit cell paramet’ers a and c were reassessed by the post-refinement procedure (Winkler et al., 1979: Rossmann et al., 1979) while keeping the mosaic spread constant at @15”. The initial cell paramet)ers of a = 142.3 and c = 104.0 .A we&a refined to 142.0 and 103.7 A. respect,ively. in space group 1’3221 for all but the HgCI, data set. The c-axis was found to be 1032 A in the latter compound showing a small departure from isomorphism. Three cycles of scale and anisotropic temperature factor adjustment followed the post-refinement of each dat,a set.. Data collection statistics are given in Table 2. r\n analysis of the completeness of the available native data is given in Table 3. Each derivative was independently scaled to the native data, evaluating mean local scale factors in 10 shells of equal volume m reciprocal space. The effective scale factor for each reflection was determined from a smoothed curve. Table 4 shows mean differences between native and taach derivative relative to error estimates as a function of resolution. It can be seen that the size of the mean differences were generally larger than the mean errors at, all resolut,ions and ti)r all compounds. t Abbreviations

used: Hg*Ac. dimercur?

acsetatr: m.i.r.. multiple isomorphous

replacement.

a* o* (I * (.* I-*

Crystal mount

168,293 119.902 91.303 47,892 45.622

Total 0bs.i (no.1 133.372 95,807 72.750 38.280 37.102

Full refl (no.) 34,921 24,095 18,553 9612 8520

where

I h is the mean intensity

of the i observations

Ihi for reflection

h

et trl. (1979)

400.624 32,538 31.742 27.906 23.132

(no.1

(no.)

collectim

ITnique refl.f,

datfl

Partial refl.f

of intensity

t Sot including overloaded reflections. $ Reflections with an estimated partiality of less than 60% are omitted (see Rossmann $ Full reflections and partial reflections with partiality greater than 60(+,. (1 Only reflections with intensities I > lu are included in the H-factor calculations.

Sative Mersalyl Ptcl, Hg& Hg(‘1,

(‘0mp011nd

Statistics

for definition

73 i3 45 83 29

overloaded (no.)

of partiality)

2.5 25 2.5 2.5 24

(4

Maximum resolution

66 58 3i 21 3I

so. of films

IO.3 9.2 8.5 9.6 9.8

Film-film scaling R-factor (00)11

3

TABLE

Suwlher

ill different

of native data available

resolution, and error ranges

lo Resolution range (A)

I

Overload

10

5

3

2

2

73

394

1098

4

85

499

1148

5

130

564

1255

1756

12

225

796

1777

1186

11

319

1056

1611

559

22

461

1455

1423

197

28

589

1661

939

67

25

789

1811

648

35

22

864

lil3

422

17

32

1001

1435

298

5

36

1041

1245

227

3

199

5579

12 ,62').

2293

10.614

ICrror rangrs are between 1 5 I/o < 2,2 5 l/o < 3..

TABLE

8200

73

97.5

0

98.7

1

96.4

0

98.2

0

940

0

92.6

0

89.9

0

87.2

0

8@3

0

73.5

0

67.1

74

88~3

., where I represents the intensity.

4

dij~~rrrrcr~s in structure amplitudes betu:een native and each derivative relative to error estimates as a function of resolu,tion

Mrun

Resolution * Illl?t-S~l~l r.m.s. delta ,‘.rn.S. erwr KzlW‘l

10%)

21.60

ranges (!I)

7.20

3.60

$32

5.40

Pi0

3.09

1x1.3

157.1

143.6

133.6

122.3

92.2

71.6

2X.6

28.3

28.4

28.8

33.5

37.0

42.7

46.X

157.3

109.7

w4

27.5

75.4 26.X

641 28.0

62.2 32.8

63.5 39.0

62.6 46.8

63.5 53-9

87% 52.5

R3.3 59.2

146-7

107.1

65.3

69.0

207%

4

,‘.m.s. delta r.m.s.

Theoretical (%)

7

r

error

H&c ,‘.m.s. delta

2804 30.2

202.1 29.7

156.6 29.2

1141

102.1

99.9

PrTOI

31.0

36.7

43.x

r.m.s. delta r.m.s. crro,

299% 32.3

234.0

200.9 32.0

174.0 37.1

2Ml 44.6

196.0 55-2

r.m.s.

Hg(‘l,

overall

of-merit

tigures-

0.78

29.3 0.81

O%O

0.79

0.72

W66

0.6 1

045

42)

M.

R.

N.

M’lrRTHY

3. Heavy-Atom

ET

AL.

Determination

The orientation of the molecule about the crystallographic S-fold axis was drt’ermined hy use of the self-rotation function using spherical co-ordinates K. 4. Q (Rossmann & Blow. 1962). The radius of integration was taken as 60 A. with dat*a limit,ed to 5 t,o 10 L& resolution using 65X large terms to represent one of t,he Patterson functions. In the light of the special molecular position. t,he anticipated to any rotation function peaks had to be confined to a great’ circle perpendicular one of t,he crystallographic 2-fold axes. The results revealed the independent molecular 2-fold axis at (CI= 90”. f$ = - 13.5” (Pig. 1). A vector search procedure similar t,o that described 1”~ Argos & Itossmann (1974,1976) was used to find the major sites of the mersalpl derivative. With knowledge of t’he molecular orient’ation. it was possible to compare t)he vectors bet)ween atoms in a molecule for a proposed at,omic position. Int,er-moleeular vectors could. however, he computed only 1)~ assuming a position of the molecular center along a crystallographic %-fold axis. Hence, the vector search was essential]> four-dimensional. alt)hough the search for the molecula,r eent’er along the 2fold axis

FIG. 1. Rotation function of beef liver catalase. Radius of integration was 60 A with data between was represented hg the 658 largest terms.

Stereographic projection of the K = 180 hemisphere. 5 and 10 ,i resolution. One of the Patterson functions

STRUCTURE

OF

BEEF

LIVER

(‘ATAL.4SE

-ii 1

was severely restricted. This position had been shown to lie between 0.56 < y < 0.72 mersalyl-natjive difference Patterson (Eventoff Pt al., 1976). A 5 A4 resolution synthesis was calculated with 5073 terms. The molecular center was assumed to be at 0. y; 516 and the search for heavy-atom positions was carried out within a sphere of radius 60 LA for each y between 0.566 and 0.726 at 0.02b intervals. The procedure yielded two pairs of independent heavy-atom sites (Fig. 2) when the molecular center was placed at y = 0.65b. A comparison of these positions (A and B) as determined by the vector search and those eventually derived from least-squares rrfinement is given in Table 5. The differences do not exceed 0.85 a. Major heavy-atom sites in the other derivatives were determined by use of single isomorphous phases from the mersalyl derivative. Some care had to be taken t’o establish the presence of A and B sites in the other derivat’ives by use of feedback experiments. The major sites of HgCl,, Hg,Ac and PtCl, were also independentI) caonfirmed by the Patterson vector search method. Minor sites were found with mult~iple isomorphous phase determinations after some init,ial refinement of heavyatom parameters at 5 A resolution. Once all sites had been determined, the heavy-atom parameters w-ere refined to 2.7 LA resolution. There was no evidence of elongation of the heavy-atom difference Freaks in the Hg,Ac derivat’ive. Presumably this compound did not. after all, contain two mercury aboms. Thus, isotropic shape parameters were used throughout the> refinement for all heavy-at’om positions. Xt,oms in the non-crystallographic 2-foldrelat.rd subunits were treated independently. The final average figure-of-merit for -P

i

R

FIG. 2. Location of methods. The orientation to locate the molecular y = M.5 corresponding molecular center. (b) I. of B site with position 16

the heavy-atom positions in of the molecule determined center and the heavy-atom to the mersalyl A site. (a) Peak at the same molecular of molecular center.

the mersalyl catalase derivative by vector search from the rotation function calculations was used positions. (a) 1. Peak in the vector search map at, 2. Variation of height of A site with position of center showing the B site. (b) 2. Variation of height

M. R. K. MlJRTHY

ET

Molecular

center

AL,

(a) 2

-P t

-0 (h)

:I

I

,,/,,,, OF56

0.58

0.60

1

0.62

0.64

Molecular

0.66 center

(b) 2 FIG. 2.

0.68

O-70

0.72

STRI’CTURE

(‘om pri.wr

of mwsalyl

OF

heavy-atom

BEEF

LIVER

TABLE

5

positions?

473

(‘ATALASE

from

vector

search

and

least-squ,urr.s

ryfirwment

AI A2 131 132

- PO599 0+)33i O.lP&! - m292.5

- CbO589 0.0323 0.1233 - 0.2924

@8849 0.9095 1.2985 I.1786

0.7 138 @5731 om30 0.5350

0.7191 0.5703 0.6843 05336

0.83 @57 0.85 0.23

om4 1 0.9095 1.2985 1.1777

t Hmvy-atom positions (z, y, 2) are referred to the same molw111ar center and expressed as fractions of a unit wil edge.

extending t,o 2.7 L%resolut’ion was 0.61. The refinement statist)ics are presented in Table 6 and the final atomic positions are shown in Table 7. Since the hea\-y-at,om posiGons were refined without imposing the non-crystallographic. symmetry, a more precise det’erminat’ion of the position of the molecular center and the molecular orientation was possible by an analysis of the heavy-atom coordinatjes. Two fjarameters were defined for this purpose. The first, yO, determines the molecula,r csenter along tjhe line 06, y, 5/6, and the second, 8, gives the inc4ination of the molecular K-axis uit’h the crystallogra,phic c-axis (Fig. 3). This implies that) a position (I’; Q. R). referred t’o the molecular co-ordinate system along the molreular clyads. is related t’o the crystallographir fract,ional coordinates (I. y. z) by : :3().%08 reflections

1’ = fr($Y;!)

~0~ e x

Q =U(-l/2) f? = u($l/:!)

--c sin 0 (z-5/6) (3)

r+wy-y,) sin 0 s

+c cos e (z-5/6).

Hence the (to-ordinat)es (x,. yI , zr) of a heavy-atom position in one subunit can be transformed to (.r;. y;, z\) by the molecular R-axis into the second subunit acaczording to : ,r; zz - (‘OS18 1’, y; = (1 -ws -’ -2

--

%9).r,/2

-o(&)

It was then necessary

-yl

-r

sin %Y(z, -5/6)/(&o/2)

-r

sin 2O(z,-.i/6)/($~~)

sin 28r,/c+~os t)o minimize

20 (z, -S/6)

+zycl

(4)

+5/6.

:

which is the sum of the squared differences between the transformed co-ordinates (.I$. ,&. t;) and the observed co-ordinates (x2, y2, z2) with respect to 0 and yO, The summation is taken over the K sets of equivalent atoms and the weights, w, were taken as their mean occupancy. The results showed that @= - 13.46” and

474

M.

A.

It.

N.

MLTRTHY

Figure-of-merit

ET

AL.

statistics Mean figure

Compound(s) Phasing

of-merit

based on 1 derivative Mers Ptrl, Hg& Hg(‘1,

Phasing

based on 2 derivatives Mers + Mers + Mers + Pt(‘l,+ Pt(‘l,+ Hg,Ac

Phasing

@50 0.60 0.35 O.‘&l 049

based on 3 derivatives Mers Mers Mers Pt(‘1,

Phasing

046

Pt<‘l, Hg,Ar HgCl, Hg,Ac HgCl, + Hg(‘l,

+ PtCl, + Hg*Ac + PtCl, + HgCl, + Hg,Ac + HgCl, + Hg,Ac + HgCl,

based on 4 derivatives Mers + PtCl,

+ Hg,Ac

0.i 1

+ Hg<‘l,

0.61

Overall+ t The number

of derivative

B. (lompound

Refinement

amplitudes

available

statistics

on heavy-atom

Mersalyl

R-modulus R-weighted Definitions

structure

27.7 1@O

for each reflection

PVl,

varied

1 to 4

compounds %-AC 335 114

87.8 58.7

from

Hg(‘1, 559

34.5

:

hmhdus

= ~~III~HI-I(~+f~lll~l~lfl

R-weighted

=

~w{~FHI-I(F+f)~}2/~wf2,

where F is native protein heavy-atom contribution

structure factor, E’, is heavy-atom and w are weights based on error

derivative estimates

of

structure factor. F, and F.

f is calculated

y0 = 0.652, corresponding to - 13.5” and 0.65 obtained from the rotation function and vector search methods, respectively. These refined values of molecular orientation and position provided a basis for comparison of the equivalent sites in non-crystallographically related subunits (Table 7). Differences never exceed 1.1 A and average to 0.6 A. Possible causes for the larger errors in the B site determination are discussed below.

45 45

46 50 25 34 16

(

A H

A R E I) F

Fwl,

Hg,Ac

W’l,

-00530 @1276 -0.1625 -09677 - 0.0449

- 0~0567 P1251

- OG44

- @0589 0.1233 - 0.0597

z

@6188 0.6035

0.7231

0.4iil

0.7191 0.6843 0.4633

?I

Subunit

pnmm,d~ra

1

0.8739 1.2994 0.5143 1.2077 1.2302

(k8762 1.3009

0.8841 1.2985 @8346

2

obtained

from

15 26 16 40 32

39 23

33

26 24 27

R

high-resolution

Occupancies, %, are on a relative scale roughly equivalent to electrons. Positions x. ,y, z are in fractions of unit cell edge, referred to the same molerular center Temperature factors R are in 8’. A is the difference in Ah between equivalent sites generated by the molecular R-axis.

29

47 46 22

A I3 (

NkW%l~l

z

Site

Compound

.-I tomic

49 44 24

47 47

25

48 48 18

z

0.0323 -@2860 0.2631

0.034 1 - 02911

PO323 - 0.2924 0.0526

s

IQast-wprrrrs

0.5676 0.5386 0.6906

0.5692 0.5345

WX267

05703 0.5336 0.8357

?I

Subunit -h

rqfirlemrrrt

2

0.8996 1.1862 0.6356

09026 1.1811

08.532

09095 1.1777 0.8672

z

12 28 5

@44 0.95 @50

0.11 0.28

012

44 31 29

0.25 w79 0.25

A 23 22 26

R

476

VI<:. 3. Ikfinition axea. n. h and c.

M.

of the molecular

K.

X.

synmetry

4. Molecular

MITRl’HY

K7’

AL

axes I’. 6, and R with

Replacement

rrspwt

to t,he crystallographic

Averaging

The multjiple isomorphous replacement electron density map was further refined by molecular replacement averaging (Buehner et ~1.. 1974: Brivogne, 1976). This. however, required an accurat’e knowledge of t,he c/a ratio (see eqn (4)). t,he ccnt,er and the molecular orient,ation in order to avoid errors in associaGon of equivalent electSron densities (Abad-Zapatero PI nl.. 1980). The c/a ratio of 0.7303 was accepted from t,he post-refinement calculations without further modification. but, 0 and y0 were det,ermined to greater acscburaq by an examination of t’he no11crystallographic symmetry present in t’he m.i.r. map. For this purpose, 60.000 point,s were selected wit,hin a molecule and the root-mean-square difference in the electron densit)y values between t,hesc points and those related 1)~ the ~OIIwere systematjically examined with respect t’o crystallographic symmetry molecular orient,ation and position. The rrsultjs are shown in Table 8 and are closely consistent, with t,he earlier values of 6’ and yO.

STRCC’TCRE

OF BEEP

LIVER

(‘ATALASE

17;

It is not necessary to select a molecular envelope for the first electron density averaging. For further molecular replacement calculations. however, it was possible to discern the molecular boundary of catalase from the averaged m.i.r. map. The envelope was examined and corrected for overlaps between neighboring molecules when placed back into the crystal cell. This final envelope was accepted without modification tjhroughout t’he molecular replacement averaging procedure using a modified version of the program package written by I)r ,J. E. ,Johnson (1978). However. the molecular orientat’ion. the position and the molecular envelope were rechecked aft)er the first cycle, but were not changed. The intervals at which the electron density was sampled along t)he crystallographic a, 1) and c axes were 0.55. O..iT, and 0.58 ,&. respectively. The solution region was set to zero for each cycle. The heavy-atom ghosts in the initial m.i.r. map were eliminated by setting the electron density \\ithin a sphere of 3.5 A radius to zero at these sites. Dat’a to only 2.7 .-i resolution were used in the first five cycles at which stage t,he data t,o 2.5 .A resolution \vere included for another tire cycles of refinement. The mean phase shift bet\veen the last two cycles was 1.1’ (Table 9), the final correlation coefficient was O.!U and the final R-factor was lci’j, (Fig. 1). The mean phase shifts from m.i.r. phases for reflections belonging to different figure-of-merit ranges follow expected behavior (Table 10). The final electron density map was far superior to the initial m .i.r. map and was easily interpretable in t.erms of the known amino acid sequence. calculations were The imJu-ovement was remarkable. particularly since averaging

Data

with

resolution

2.X

I .2

25

1.4

2.7

1.i

2.6

1+s

2.8

19

3.0

2.0

34

2.2

593

2.6

11.1

39

15.1

44

bet,ter than 2.7 .k were int,rodured after the fifth cycle of ralrulat~ions.

4ix

M.

R.

9.

MI’RTHY

EC”

j-

, 0

0.05

Resolution 1 0.10

AL.

---13.3

\ 2.5 A

L o-15

, 0.20

(8) ,

40

30 I 20 1 d

0

Ilb)

1 IO

.-.-I~~

----A 5

Resolution

3.3

/ 2.5

(&I

Plc:. 4. Variation of correlation coefficient (a) and K-factor (h) with stages of molecular replacement calculations. (See Abad-Zapatero definitions of correlation coefficient and R-factor.)

respect to resolution at different et ul. (1981). for instance. for

only over two subunits. The excellent quality of the final map is demonstrated Figure 5 show’ing part of t,he heme environment.

5. Description

and Overall

Organization

in

of the Molecule

The molecule is roughly dumbbell-shaped. The tetramer is 105 w along the P and R-axes, exhibits a waist of 50 a in the R = 0 plane (Fig. 6) and is in reasonable agreement with the electron imaging results of Barynin et al. (1979). Identity of the

STRITC’TI:RE

Ijiwrq~nw

Kgure-ofmerit (m)

of

phases

from

OF

BEEF

LIVER

TABLE

10 dur values culculations

their initial rrplawmrn,t (‘Jdr

1

The right-hand cwlurnn shows co-’ give-n figure-of-merit range.

to

47!)

rqfinement

hy

molwular

no.

4

m, which

(‘ATALASE

10

7

is a measure

of the standard

C”S

error for reflections

’ m

for the

PIG. 5. The quality of the final map is represented in a stereo view of part of the heme environment. The proximal ligand is Tyr357, whereas His74 and Asn147 are on the distal side and are involved in substrate binding with the heme.

480

M.

FIG:. 6. Overall shape of the catalasr the buried heme groups.

K.

N.

MCTRTHY

molecule

showing

ICY’

AL

t,he approximate

location

and orientation

of

space groups in which beef liver and P. vitalr catalase crystallize together with their nearly eyual a-axial lengths of 142 and 145 8. respectively, suggest that the same molecular 2-fold axis is incorporated into the crystal symmetry in both cases. rls far as can be ascertained from published informat.ion (Vainshtein et al.. 1980). t.his is indeed the situation. The four heme groups are well buried in the tetramer, at) a dist’ance of 20 Lh below the molecular surface and 23 AA away from the molecular center. It could be easily recognized in the m.i.r. map as a flattened densi@, 38?,, higher t,han t.he largest protein density. The relative height of the heme iron peak with respect t’o the average protein value remained constant during the molecular replacement calculations. The heme iron at,oms related by molecular P, & and Raxes are at’ a distance of 30%,453 and 34.5 .A. respectively. The heme normal makes a small angle of 19” t,o the molecular P-axis. A scaled model of the molecule was const,ructed in a Richards (196X) optical comparator. The st,ructure was in complet,e agreement) with the amino arid sequence (Schroeder et al., 1969: W. A. Schroeder, personal communication), with most side-chains being clearly visible. However. the four amino-terminal residues. six carboxy-terminal residues and residues 18 t,o 21 had no appreciable densit,y. For a small number of residues (Glu85; HislOl, ArglO5. Ala288, Glu289, Arg379. Met394. Gln414, Arg421) on the surface of the molecule, side-chain densit’y was lost as these were found to have been truncated by the assumed molecular envelope. The density corresponding to the external parts of the polypeptide chain t,hat do not belong to the internal eight.-stranded p-barrel domain (vide in@) was of somewhat lower quality. C, co-ordinat,es have been deposited with the Brookhaven Protein Data Bank and a full set of co-ordinates will be submitted in due course. The secondary structure of catalase is represented as a backbone hydrogenbonding network in Figure 7, as a stereodiagram of the C, atoms (Fig. 8(a)) and a diagrammatic representation in Figure 8(b). According to usual convent.ions: nhelices have been numbered 1 through 13 and strands within p-sheets 1 through 9. Helices account’ for 269; and sheet residues for 120/;, of the molecule. Optical rotatory dispersion measurements had indicated around 500/(,,helical content (Yang

STRUCTURE

OF BEEF

LIVER

(‘ATALASE

4x1

ot Samejima. 1963). The st’ructure of each subunit can be considered as being c-omposed of four structural domains. The first domain (Fig. 9) comprises t,he amino-t,erminal 75 residues, which form an arm ext,rnding from the globular region of each subunit. The arm consists of t,wo helices (11 a,nd 12) both of which are involved in intersubunit contacts, the first \vit.h the Q-axis-related subunit and the second with the essential helix and heme of the K-axis-rrlat,ed subunit,. Other amino-terminal arms have been implicated in the a.ssrml)ly of Iact,ate dehydrogenase (Adams rt crl., 1970) and of spherical viruses (Harrison rt nl.. 1978: Abad-Zapatero rt al., 1980). Thr~ sec~~nd domain (Fig. 10) comprises residues 76 t,o 320, which form a large eight-stranded anti-parallel sheet p-barrel. The heme’s distal side int,eracts with the exterior of this barrel. The p-barrel constitutes two, topologically similar (pl to p4 and /35 to fi8). four-stranded. anti-parallel sheets such as occur in rubredoxin (K’atenpaugh e/ (11.. 1979). Most residues on the dist,al side of the heme (I’a173, Hisi-1, \‘a11 15, Phel60, Phel52 and Asnl-17) originate from this region of the molec~ul(~. The stret)ch of polypeptide chain between t)he topologically similar halves of’thcb p-barrel cdontains three helicaes (13 to n5) and contributes to a hydrophobic2 csnvironment on t,he distal side of the heme. l’hca third domain (Fig. 11). residues 321 to 436 to be referred to as the “wrapping domain”. finms an outer layer to each subunit. This does not have obvious similarities to other previously observed structures. It lacks discernible secondary struc+ure in a long stretch of polypeptide chain between residues 366 and 420. However. this tlomain contains the essent,ial helix (19) with t’he proximal ligand Tyr3.7. The wrapping domain also forms a short p-st,ructure (/39) with an identiral region of thcx Paxis-related subunit. The carbox),-terminal portion of the molecule (residues 137 to 506) is folded into a fom-hrlit*at domain (210, 11 1. 11%. 113) with a topology and hand similar t,o the hrtic,es E. IT. (: and H of the gtobin fold (Fig. 12). This domain is removed from the active site a,nd is not involved in intersubunit interactions. However, it limits the at~c~t~ssibility of the actjive caent)er by contribut,ing t,o t,he formation of the hydrophobic c*hannel leading to the heme. ‘I’htb stru(+ure of beef liver cat)alase reported in this paper is similar to I’. /*itnIP c,at alast, (\~ainshtcin rt al., 1980), although it lacks the “flavodoxin”-like domain. found a.t the t*arboxy-terminal region of t,hc latt,er. indicating the conservation of a, basics catalasr strucature during evolution. The additional domain in P. &C//P c.atatasc, might indicate an additional nut-leotide binding function of t,his molecule (Rossmann rf (I/.. 1971). The obvious conservation of t,he basic csatalase fold argues strongly in favor of divergent evolution of this molecaulr from at least, earl! clukaryotic organisms. The extra domain in fungat caatalase may be the result of gent’ fusion with a nucleotide binding gene and would then be another example of t tltl sc>parat (’ evolutionary development of func+ional prnt,ein domains (Itossmanri & Liljas. 1971). possibly supported by gene organization into coding and non-coding sequt~nc~es (see .Irt,ymiuk et al.. 1981 ). .\n int rasubunit diagonal distance plot (Phillips, 1970: Rossmann B Liljas, 1971) shou-s th(l tlomain structure and the domain-domain interactions of beef liver c*atatatitl (I:ig. 13). The ext)ended feature of the amino-terminal arm is reflect’cd in

a5

a3

a2

N 0

M. R. N. MI’RTHY

E?

AI,

(b) Flc:. 8. (a) &ha carbon backbone of one catatase subunit. The heme group is shown with thicker bonds. (b) Diagrammatic representation of serondary structural elements.

STRI’C’TURE

OF

BEEF

LIVER

CATALASE

48.5

R

FIG:. 9. Organization

//” \

of the amino-terminal

arm within

the tetramer

, ‘, -.

FIG:. IO. Arrangement of the g-stranded ~-barrels the residues on the distal side of the heme.

within

the tetramer.

The p-barrels

provide

most of

48fi

M.

R.

N.

ML’RTHY

ET

AL

R

FIG. 11. Structure of the 4 wrapping domains in one catalase molecule. layer in each subunit. The essential residue Tyr357 is contained in helix

These domains form n9 of this domain.

an outer

l-

PIG. 12. Organization restricts the accessibility

of the carhoxy-terminal of the heme groups.

helical

domain

in the tetrameric

molecule.

This domain

~Amino-termmol arm i

Borreldomotr

I

Wrapping domain

Fo;lr IWccll domwh

STRUCTURE

OF BEEF

LIVER

CATALASE

487

t,he absence of interactions away from the diagonal for residues 10 to 72. However, due t,o a p-bend in the arm, the first nine residues interact with the p-barrel domain of the same subunit. The plot between residues 76 and 320, constituting the fibarrel, shows a large number of interactions away from the diagonal as a result of the compact, nat,ure of this domain. Furthermore, this part of the plot shows a repeating pattern corresponding to residues 76 to 150 and 200 to 320 of the p-barrel. The interactions of the wrapping domain between residues 321 and 436, which are distant from the diagonal, correspond to non-specific hydrophobic interactions of this domain with the /?-barrel domain. The compact carboxy-terminal domain shows only a few contacts with the rest of the subunit. The relative substitutions of t’he heavy-atom compounds used for the isomorphous replacement method and the amino acids involved in their binding are shown in Table 11. The A and B sites are located at the exposed sulfhydryl residues, TABLE

il mino acids involved

11

in binding

heavy-atom

Site

Average 0wupanc.y

Residue

Subunit(s)

A B (’ E 1) F

47 46 22 25 34 16

(‘ys392 (‘ys459 Cys376 His234 His254 Glu255

1,2 I,2

I,2 1.2

l-2 1

compounds

Compound(s) Mers, Mers, Mew, Wl, HgCI, Hg%

Hg,Ac, HgCl, Hg,Ac, HgC& PtCI, (on R-axis)

(‘ys392 and Cys4.59, respectively. The C site, which is also at a cysteine residue (376). is not fully substituted presumably because it is partially buried. However, only Cys376 is reactive with respect to PtCl,. Cys231, which is totally buried, does not react with any of the heavy-atom compounds used in this study. The minor site E, found only in the HgCl, derivative, is associated with His234. The D site is very close to the molecular R-axis and is associated with His254, probably from both subunit’s. The F sit’e is 6 A away from the R-axis and relates to Glu255 in only two of the four subunits. The polypeptide chain exhibits least conformational flexibility in the b-barrel where the electron density is crispest. The difficulties encountered while interpreting other regions in the initial m.i.r. electron density map and the disorder observed at residues 18 to 21, the carboxy and the amino termini indicate greater flexibilky in the wrapping domain as well as disorder at the chain termini. The larger error in the molecular symmetry of the heavy-atom B sites (Table 7), in the four-helical domains, also reflects the greater flexibility of these domains. Most of the charged residues are found on the outside of the molecule. Residues have been roughly designated as “external” or “internal” in Table 12. All the internal charged residues, except for Lys236, are either charge-balanced or occur in hydrophilic environments (Table 13). Lys236 is a most unusual residue occurring in a hydrophobic environment in the interior of the p-barrel domain. 17

488

M. R. N. MURTHY TABLE

External

and internal

ASIl

ASP QS

Gin GlU Gly His Ilr Leu L.YS Met Pro SW Thr Tyr \‘a1

224 18 421 39 480 9 248 392 17 16 452 103 13 102 175 12 475 391 6 8 87 83 246

269 46 443 223 500 24 256 459 21 66 453 117 88 241 447 15 476 393 23 119 91 93

288 92 455 243

380 105 457 266

Arg Am Asp cys Gin GlLl Gly His Ik LW

LYS Met Phe PI.0 Her Thr TOP TY~ Val

61 326 71 141 53 231 52 59 29 213 74 90 26 261 466 37 11 56 199 481 33 340 40 27 14 136 25 145 449

12

Exposed

catalase molecule

residu.es

400 410 417 463 469 494 126 169 188 202 209 251 262 319 379 381 491 272 294 318 320 396 432 435 438 451 461

36 89 127 139 177 179 183 201 206 212 225 238 258 263 306 347 388 395 427 436 437 468 483 497 167 85 460 120 101 310 471 22 479 394 107 121 173 214

172 100 487 226 174 95 498 292 186 237 230

239 281 371 397 414 429 441 454 470 474 493 118 138 227 247 255 287 289 327 412 419 240 271 305 398 399 426 464 210 234 254 304 413 420 423 465 485 97 104 168 176 220 242 314 448 456 467 308 245 270 259

311 275 277 273

B. Ala

AL.

residues in the tetrameric A.

Ala A%

ET

415 286 284 279

486 407 416 425 433 482 490 422 440 444 307 378 404 446 499

Buried

residues

75 332 129 147 64

78 356 353 148 143

80 383 362 323 156

96 109 116 132 157 228 249 250 253 267 477 364 337 368 375 297 334 359

330 329 30 215 208 108 38 264 496 134 60 63 219

351 343 31 260 217 151 41 278

35 313 361 154 49 298

44 341 363 164 50 309

48 344 113 28 142 235 34 195 478

128 346 162 42 182 324 43 222 484

47 77 79 82 130 140 146 203 207 352 366 389 489 204 268 280 290 342 372 462 473 492 144 158 159 187 192 198 221 244 252 315 317 331 350 354 365 370 450 458

232 236 348 180 211 283 338 349 81 84 99 112 131 135 152 153 160 163 184 197 233 265 285 291 293 296 325 333 355 408 424 445 150 358 196 57 185 357 51 229

161 367 200 106 276 369 54 282

171 373 216 114 302 403 55 301

205 377 336 124

257 390 345 137

274 295 303 321 335 339 401 411 409 149 218 299 360

488 72 73 86 98 110 115 125 133 312 316 322 328 374 382 428 442

STRUCTURE

OF BEEF TABLE

Ionic

interactions

LIVER

Asp127 Asp123

Argl29 Lys134 Asp143 Argl55 Asp156 Lysl68 l&76 Glul90 Arg209 Asp256 Asp258 Arg262 Asp263 Asp297 Lys3OO Gln329 Asp334 I A!,7.948 h. Asp359 Arg362 Arg364 Arg381 (:lu419 Arg430 .4sp436 (:lu452 Glu4.53 Arg4.55 I &56 .hg457 Glu487 .hp497 I+498

489

13 within

a molecule

Charged residue in the reference subunit Asp9 Lys12 Asp24 Asp36 Lys37 Asp53 Glu59 Asp64 Arg65 Glu66 A@7 Arg7 1 Lys76 Arglll Glul18

CATALASE

Charged residues within 6 A LYSl2 Asp9 A@81

(‘2)

Arg430(Cl) Glu59 4rg430 (R) Lys37 Arg65, Asp359 (R) Asp64,Asp359 (R) Arg67, Lys168 (R) Lys168 (R), Glu118, Glu66 Arglll, Glu329, heme propionyl, Arg364 Asp123, Asp258 Arg71, Glu329, heme propionyl Arg67, Lysl68 Arg129 Lys76, A@29 Asp123, Asp127 Asp1 43. Asp334 Lys134. Asp334 Asp297, Glu190, Lys300, Asp436 Lys348 Arg67 (R), Glu118, Glu66 (R) Asp256 (R), Asp258 (R). kg262 (R) Argl5.5 Asp263

Lys176 (R), Arg262 Lys76. Lysl76 (R) Lys176 (R), 4~~256 Arg209 Argl55. Lys300 4sp297. Arg1.55, Asp436 Brg71, Arglll Lysl34, ,4spl43 Asp156 Asp64 (R). Arg65 (R). A@62 Asp359 Arg71, heme propionyl Asp24 cc9 Arg430 (P)

Asp36 (Q), Asp53 (Et), Arg430 (P), Glu419 (p) Agl55, Lys300 Lys456 Lys456, Arg457 Oh1487 Glu453. Glu452 Glu453 Arg455 Lys498 Asp497

Residues on neighboring subunits are designated with (P), (&) or (R) according to the relationship the residues in the reference subunit shown in the left-hand column.

to

490

M. R. N. MURTHY

ET

AI,.

6. Quaternary Structure The subunit contacts generated by the molecular I’, & and R-axes of catalase arc illustrated by an interaction tetrahedron (Rossmann et al., 1973) in Figure 14. Most of the intersubunit contacts are confined t,o the amino-terminal arm and t,he wrapping domain. The first sectSion of t)he arm (residues 15 to 35) is in contact with the &-axis-related subunit while the second se&on (residues 36 to 73) interacts with the R-axis-related subunit. Due to a change in direction between helices 11 and a2 the actual amino terminus is situat’ed not far from the p-barrel domain of the same subunit (Fig. 8). The first, 25 residues of t,he arm are inserted into a hole generat’ed by excursion of residues 390 and 420 and by residues 138 to 141 1 337 t)o 340 and 378 to 420 of the &-axis-related subunit’ (Fig. 15). Arg71 on the arm interacts with a propionic acid side-chain of a heme belonging to the same subunit. Residues 421 to 426 in the wrapping domain form an anti-parallel /3-sheet’ wit*h the same secondary structure feature across the molecular P-axis. Similar situations exist in superoxide dismutase (Richardson it nl.. 1975) and liver alcohol generates t,wo fourdehydrogenase (BrB;ndBn et al., 1975). The molecular symmetry helical bundles, each helix belonging to a different subunit, parallel to the molecular P-axis. The heme groups are on the outside of this bundle (Fig. 16). Tetrameric catalase is normally isolated as a holoenzyme, even when monomers are produced in excess of available heme groups (Ruis, 1979). Hence. it) was concluded that it’ is only possible to form tet,ramers with holo subunits. It has also been shown that acid denaturation (Samejima Kr Yang, 1963) or lyophilizat’ion (Tanford &r Lovrien, 1962) produce only a highly ellipsoidal single dimer species. From these observations it) can be concluded t,hat the biosynthesis of catalase involves the following steps : (1)

M Monomer

+

H Hemc

-+

MH Ho10 monomer

(2)

MH

+

MH

+

WH), Ho10 dimer

(3)

OfHI,

+

(MH),

+

(MH), Ho10 tetramer.

The structure supports these findings. No essential difficulty would be experienced in making a P-axis dimer. However, assuming the same structure for t’he loose wrapping domains in the P-axis dimers as exists in the assembled tetramers, it’ is unlikely that the resultant dimers could assemble int,o tetramers because they would have to insert the 68 amino-terminal residues through a 32 A channel along the molecular P-axis. Formation of tetramers via Q-axis dimers is also improbable as that would require the threading of the amino-terminal arm through the hole mentioned above. Only t)he R-axis dimers present themselves as viable intermediates in tetramer formation, as the requirement for intricate navigation of the amino-terminal arms is absent. Furthermore, these dimers are the most consistent with the results of Tanford & ellipsoidal of the three possibilities, Lovrien (1962).

NC:. 14. IIiagramrnatic repwsmtatiorr of contacts betawn residues in neighboring subunits. The reference subunit is represented by a triangle with its edges wpwsenting the 3 difftwnt interactions generated hy the molecular I’. Q. K-axes. Shown are all (‘,A ‘I distances less than 5 .%.although contacts between Rl-51, IHP-NX3. 171~~3RX.171-399 grneratpd by the Faxis and betwren 31-110 32~140. W-398. 3% 396 generated by th? Q-axis have been omittrd for &wit>

492

H

M. R. N. MLTRTHY

E7’

AL

R-

FIG:. 15. Thr first 25 residues of the arm in one subunit are inserted int,o an opening of another subunit related t,o the first by t,he molecular Q-axis.

P

FIG:. 16. Interaction of helicrs running parallel to the molecular I’-axis. The heme groups are on the outside of this helical bundle.

STRUCTURE

OF BEEF

LIVER

CATALASE

493

Acatalasemia is known to occur in patients with a mutant catalase in which the tet,ramer has the same specific activity but there is a tendency to dissociate into dimers of low a&vity (Aebi et al., 1974). However, t.he mutation sites remain unknown. A mutation that would favor the formation of P-axis dimers. in preference to the productive R-axis dimers, would hinder tetramer formation and leave the heme partially exposed, thus drastically altering act,ivity.

7. Heme Environment The heme site is accessible by a channel 30 A long and 15 A wide that occurs between the /!-barrel and the carboxy-terminal domains. The channel opens toward the molecular R-axis and is lined with the polar residues Arg126, Asp127, Gln167, Lysl68 and Lys176 at the entrance and with the hydrophobic residues Vall15, Alallfi. Pro128, PheI52, Phe153. Phe163, Ile164 and Leu198 as the channel descends t.oward t,he heme, narrowing as the heme is approached (Fig. 17). The limited diameter of this channel can account. in part, for the reduction of the rate of oxidation by about an order of magnitude for each additional carbon in the K. group of straight-chain alcohols (Chance, 1949). The hydrophobicity of the channel interior can readily explain the requirement, for the neutral species in ligandexchange reactions (Chance, 1952). The heme Fe co-ordinates in the reference subunit are 1634, 3.38, and 1603 a and the heme normal has direction cosines of -0.321, -0.718 and @617 with

VII:. Ii. writer.

Viw

of the hydrophobic

channel through which the substrates must diffuse t’o t,he active

494

M. R. N. MURTHY

ET

AL.

respect to the molecular P, & and R-axes. The heme handedness. which is determined by the asymmetric distribution of propionic and vinyl side-chains, was easily identified (Fig. I$). The essential residues within the heme pocket differ substantially from that, of other known hemoprotein structures (Fig. 19). The heme cavity is essentially hydrophobic wit>h int,erspersed hydrophilic residues. On separation of the tet,ramer into monomers. pyrrole rings 1 and IV become partiall,v exposed (Fig. 8). The heme edge formed by pyrrole rings 11 and III abuts on strands p2, /33 and 84 of the p-barrel which supports the heme. The faces of t,he heme are surrounded by helices. Both propionyl groups are directed toward the molecular center and, unlike most other hemoproteins, are buried. The propionic acid of pyrrole ring III is neutralized by the guanidinium group of Arg71 while t,hat of pyrrole IV is neutralized by Arg364. Of all the other known hemoprottin structures, only the globins form protein-propionyl salt,-bridges (Watson, 1969). The heme vinyl and methyl groups interact by van der Waals’ contacts wit,h norl-

FIG. 18. Superposition of heme structure on electron density. and its orientation. The view is from the distal side.

cl&lg

identifying

FIG. 19. Essential residues in the heme environment

hand of the herne

STRUCTURE

OF BEEF

LIVER

CATALAXE

405

polar side-chains of the protein. A list of heme side-chain-protein interactions is given in Table 14. The distance of the Fe atom in the reference molecule to each of the (‘, positions in all subunits is shown in Figure 20.

TARLE 14 Humr-protein

interactions

Side group

Contacts

I

Methyl Vinyl

MetGOt, Phel60 Ala1.57, Phel60, M&349. Gly352

II

Methyl Vinyl

Asn147. Phe1.52 Gly130, Glyl46

III

Methyl Propionir acid

SerllY, Gly130. Ala132 Arg71, His74. Argl 11. Set-113

I\.

Methyl Propionic acid

Phe637, Asp64t, Thy360 Thr360, Va172. Arg364. His361

Pk~role

ring

t Residur from the R-axis-related

subunit,

The proximal and distal heme sides can be readily identified. Tyr357 provides a protein ligand (Fe-phenolic oxygen distance is 1.9 A) on the proximal side, while there is no protein ligand on the opposite side. Furthermore, His74 is located near the latter side. Modification of this residue by 3-amino-1,2,4-triazole (Margoliash & Novogrodsky. 1958; Agrawal et al., 1970) inhibits the enzyme by hindering the substrate binding. The proximal side of the heme is crowded and contains residues \‘a1145. His21 1, Pro235, Arg353, Ala356 (Fig. 21). The phenolic hydroxyl group of the proximal Tyr357 is presumably deprotonated due to the electron-withdrawing power of Fe which, although formally in the ferric oxidation state, has an effective charge of + 1. However, the guanidinium group of .4rg353 is only 3.5 a from the will favor phenolic oxygen of Tyr357 (Reid et al., 1981) and, therefore, probably ionization of the t,yrosine OH and lower its pK. This juxtaposition may also be rrsponsiblr for t,he modified spectral behavior of the heme Fe in catalase as compared to model porphyrin complexes with anionic axial ligands (Mincey 8 Traylor, 1979). Interestingly, His217, which had been suggested as a possible proximal ligand on the basis of sequence similarities between catalase and other hemoproteins (Schonbaum & Chance, 1976), is only 12.5 A from the heme Fe on its proximal side. However, it is improbable that this residue participates in catalysis. The residue following Tyr3.57 is Pro358, which does not’ terminate but redirects helix 19. In the cytochrome b, family (Guiard bi Lederer, 1979), one of the two Feco-ordinating hi&dines and an immediately following proline residue are in a conserved tetrapeptide. The occurrence of a proline adjacent to such a crit,ical rcsidur as a heme ligand raises the possibility that. the former might) play a role in t,he orientation of t,he ligand in both cytochrome h, and catalase. The distal side of the heme is much less confined and includes residues His74.

M. R. S. MURTHY

496

dl Reference

,! P-axis

E’T

AL

subunit

related

Residue

number

Residue

number

subunit

Residue

number

FIG. 20. Distance bet,ween the heme iron atom in the reference subunit and every (‘@atom in each subunit. The secondary structural elements are shown for each polypeptide.

STRUCTURE

OF BEEF

LIVER

CATALASE

497

RRG I v PHE

FIG. 21. The hemr pocket

jTall 15, Asn1-17, Phe1.52 and Phel60, but the L, site is unoccupied. In contrast, this position is occupied by a water molecule in metmyoglobin (Takano, 1977) and in cytochrome c peroxidase (Poulos et aZ., 1980). These observations can be correlated with the lack of pH dependence of the ultraviolet and visible spectra in catalase in chontrast to the other heme proteins. A small peak of electron density between His74 and Asn147 suggests a water molecule hydrogen-bonded to these residues. The oxygen of this putative water molecule is 3.8 ,& from the Fe and, therefore, is too far away to co-ordinat’e to the Fe atom. The N, of His74 is 4.3 A from the heme iron. The catalytics importance of this residue has been indicated by the loss of enzyme ac+vity and ligand-exchange capacity after its irreversible modification with 3amino-l .2.4-triazole (Margoliash & Novogrodsky, 1958: Agrawal et cd., 1970). The Fe, His74 and Asn147, then, are the essential active-site components participating in the catalytic mechanism (Fig. 19). In addition, Vail 15 and Phe152 are positioned to direct the stereospecific abstraction of the pro-R hydrogen of ethanol by compound I (Gang et al., 1973: Corral et al., 1974). The phenyl ring of Phel60 is 3.5 A above and parallel to pyrrole ring I. In cytochrome c peroxidase there is a tryptophan situated similarly above pyrrole ring II. These aromatic residues are orient,ed to form 71-n interactions with the heme. This situation does not occur in the globins. A proposed catalytic mechanism in the light of the structure and the wealth of chemical information on catalase will be presented in a subsequent paper (Reid, Rlurthy K: Rossmann. unpublished data). 1Yr &I-Pgratef’ul for assistance over a short period of time in the early stages of this project to Dr N’. Donald L. Musick. We greatly appreciated a diagram of the chain tracing of I’enidium z,itnlu catalase from Professor B. K. Veinshtein at a time when we had not yet been allIe t,o trace the complete beef liver catalase polypeptide chain. We wish to thank MS Sharon LVilder. Kathy Shuster, Peggy Baker and Mr William Boyle for help in the preparation of this manuscript. The work was supported by t)he National Institutes of Health (grant no. GM10704) and the National Science Foundation (grant no. PCM78-16584). One author (T.J.R.) was supported by a National Institutes of Health Cellular and Molecular Biology Grant and another (A.S.) by a National Institutes of Health Postdoct,oral Fellowship (no. 1 F32 GMO6364) during part of this work.

49x

M. R. N. MlTKTHY

h’T

AI,

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Edited by R. Huber