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Complexes of bacterial catalase with peroxide, azide and carbon monoxide
132
A. MUHAMMED
5 A. KORNBERG, S. R. I~ORNBERG AND E. S. SIMMS, Biochim. Biophys. Acta, 20 (1956) 215. 6 S. R. KORNBERG, Biochim. Biophys. Acta, 26 (1957) 294. F. G. WINDER AND J. M. DENNEN¥, Nature, 175 (1955) 636. 8 F. G. WINDER AND J. M. DENNENY, J. Gen. Microbiol., 17 (1957) 573. 9 0 . HOFFMANN-OSTENHOF AND L. SLECHTA, Proc. 2nd. Intern. Syrup. on Enzyme Chem., Japan, (1957) 18o. 10 A. MUHAMMED, A. RODGERS AND D. E. MUGHES, J. Gen. Microbiol., 20 (1959) 482. 11 D. E. MOGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. 12 I. A. ROSE, M. GRUNBERG-MANAGO,S. R. KOREY AND S. OCHOA,J. Biol. Chem., 211 (1954) 737. 13 A. W. D. AVlSON, J. Chem. Soc., (1955) 732. 14 F. LIPPMAN AND L. C. TUTTLE, J.Biol. Chem., 159 (1945) 21. 15 I. BERENBLUM AND E. B. CHAIN, Biochem. J., 32 (1938) 295. is H. A. KREBS AND R. HEMS, Biochim. Biophys. Acta, 12 (1953) 172. 17 G. G. BERG, Anal. Chem., 3 ° (1958) 213. 18 C. S. HANES AND F. A. ISHERWOOD, Nature, 164 (1949) 11o7. 19 W. J. NICKERSON, Experientia, 5 (1949) 202. 2o E. BUKHOVlCH AND A. N. BELOZERSK¥, J. Biochem. (U.S.S.R.), 23 (1958) 238. 31 S. SPIEGELMAN, in W. D. McELROY AND ]3. GLASS, The Chemical Basis o[ Heredity, The J o h n s H o p k i n s Press, p. 232. 22 ~¢1. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Biochim. Biophys. Acta, 20 (1956) 269. 23 S. E. BRESSLER AND KH. M. RUBINA, Ann. Rev. Biochem., (1955) 628. 24 A. A. HAKIM, Enzymologia, 19 26 (1958) 96.
Biochim. Biophys. Acta, 54 (1961) 121-132
COMPLEXES OF BACTERIAL CATALASE WITH PEROXIDE,
AZIDE
AND CARBON MONOXIDE A. C. M A E H L Y
E. R. Johnson Foundation/or Medical Physics, University o/Pennsylvania, Philadelphia, Pa. (U.S.A.) and Government Laboratory/or Forensic Chemistry, Stockholm (Sweden) * (Received April I7th, 1961)
SUMMARY
Using crystalline bacterial catalase, the spectra of peroxide- and peroxide-azide complexes were recorded at pH 5-4 and 7.0. The kinetics of the formation of the carbon monoxide compound of one of these, and its reversible light dissociation was measured. By repeating this reversible process at a series of wavelengths, a light dissociation spectrum was obtained which proved to be identical with the difference spectrum between Cat-Az II and Cat-Az II-CO. Catalase-peroxide complex III has a spectrum very similiar to that of Cat-Az II at pH 7.0. However, by lowering the pH to 5.4, the peak of complex III is unchanged whereas the peak of Cat-Az II moves from 416 m/~ to 412 m/z. Complex III did not bind CO under the conditions of these experiments. The data indicate that Cat-Az II and complex III are not identical. Quantitative spectrophotometric data on catalase from liver, blood and bacteria, as well as on the complexes mentioned above, are listed. They are compared to earlier data in the literature. N o n - s t a n d a r d a b b r e v i a t i o n s : HLC, horse liver catalase; HBC, horse blood catalase; BMC, bacterial micrococcus catalase. * Mailing address: G o v e r n m e n t L a b o r a t o r y for Forensic Chemistry, Tomtebodaviigen 3o, Stockholm 60, Sweden.
Biochim. Biophys. Acta, 54 (1961) 132-144
COMPLEXES OF BACTERIAL CATALASE
133
INTRODUCTION
OGURA et al. 1-s have shown that catalase with a hydro-peroxide (H,O2, CH3OOH, C,HsOH ) and azide forms two distinct complexes which he calls Cat-Az I and Cat-Az II. The latter was shown to be identical with the catalase-azide-H,O 2 complex first described b y KEIL1N AND HARTREE4. The spectrum of Cat-Az I I in the visible region was found to be so closely similar to that of c a t a l a s e - H 2 0 , - c o m plex I I I , also discovered b y KEILIN AND HARTREE s, that the question arises if these two complexes are identical as well. The present paper reports spectrophotometric and light dissociation data which, it is hoped, will help to clear up this question. I t is well known that HLC contains varying amounts of bile pigment and that HBC is hard to rid of traces of hemoglobin. BMC seems to have neither of these disadvantages: I t has the full complement of 4 hemingroups (like HBC), and has not been shown to be accompanied b y heroin impurities. For this reason, BMC was selected for the present studies. EXPERIMENTAL
The HBC used for the spectrophotometric data presented in Tables I - I I I was prepared in collaboration with Dr. W. J. STEELE according to the method of HERBERT A N D PINSENTs. After several recrystallizations, a RZ ( A i o s / A 28o) of 1.21 was obtained. The Kat.f. was 60000. Micrococcus L was grown according to BEERS7. and the enzyme (BMC) prepared and crystallized b y a modification s of the method of HERBERT AND PINSENT9. The preparation employed here had a RZ of 0.78. Two different recording spectrophotometers were used in this work. Spectra of all compounds described were recorded on a chopped beam spectrophotometer devised b y YANG AND LEGALLAIS10,11, either at low speed (approx. 30 m/,/min) or, if the compounds were unstable, at high speeds of about 2-6 mF/sec. "Absolute" spectra (enzyme vs. buffer) as well as "difference" spectra (enzyme vs. enzyme + reactant) were recorded. A double beam instrument designed by CHANCETM was used for studying 100 5543
~ 2o ~
o
350
400
450
(m.u)
500
550
.
600
Fig. i. The t r a n s m i s s i o n of the filters used for the m e a s u r e m e n t of the kinetics of the light dissociation. Shaded areas: light. The illuminating light is cut off below 55o m/~, the p h o t o m u l t i p l i e r receives light only in the Soret region. Abscissa, w a v e length in m/~; ordinate, t r a n s m i s s o n in percent. The curves are replotted from the suppliers catalogue (Corning Glass Works, Coming, New Hampshire). * The a u t h o r is v e r y grateful to Dr. R. F. BEERS, Jr. for detailed instructions on the t e c h n i q u e s used for growing the organism.
Biochim. Biophys. Acta, 54 (1961) 132-144
134
A.C. MAEHLY
the formation and the reversible dissociation by light of the carbon monoxide complex of Cat-Az n . This apparatus measures the difference of absorbancy at two freely chosen wave lengths as a function of time. For the study of the light dissociation, light from a Ioo-W projector bulb operated on direct current was allowed to fall on the cuvet at right angles to that of the measuring beams. The absorbancies were recorded with the help of the amplified alternating current output of a photomultiplier tube. The technique employed is essentially that of CHANCE, SMITH AND CASTOR13, except that the photocell was replaced b y a head-on type photomultiplier tube. A Coming Filter No. 3482 w a s used at the entrance opening (illuminating light), a No. 5543 filter was inserted between sample and photomultiplier. The filter arrangement is shown on Fig. i. In both instruments, cooling was provided b y an alcoholwater b a t h at - - i o ° and a pump. The temperature in the I-cm cuvets could thus be kept a + I ± 2 °. For forming CO compounds, CO gas from a tank was passed through the solutions and the cuvet closed with a greased cover immediately afterwards. RESULTS
Absorption spectra In order to obtain the absorption spectra of free catalase (BMC), of MeOOH complex I I {BMC-MeOOH II), of H20 2 complex I I I (BMC I I I ) of peroxide-azide complex I I (BMC-Az II), and finally, of the CO compound (BMC-Az II-CO) found b y KEILIN AND HARTREE 4, the chopped beam recorder of YANG AND LEGALLAIS w a s used. In a typical experiment, the sequence of reactions and the concentrations of the reactants were the following: BMC + MeOOH (0. 3/~M) (I mM)
\
BMC-MeOOH II + H,O, (0. 3/zM)
(5.0.4 mM)
BMC-MeOOH II + NaN s (0.3/~M) (5 mM)
BMC-Az II + CO (o-3/tM)
25°\
BMC-MeOOH II
(I)
~ I°\
BMC III
(2a)
~ I°\
BMC-Az II
(2b)
~ I°\
BMC-Az II-CO
\ \
(3)
2o m M phosphate buffer was used throughout, since the work of AGNER AND THEORELL14 and of CHANCE15 had shown that catalase binds m a n y ions strongly but phosphate ions only to a minor degree (pK'Fe, I-I,PO,, ---- 1.5, (see ref. 14). The concentration of catalase never exceeded 0.5 ~ M and was usually kept lower, since it was found that the higher the concentration of catalase, the more H20 , was needed to shift the equilibrium of reaction (2a) to the right. Complex I I was formed at room temperature in order to let the reaction go to completion within reasonable time. The solution was then cooled to + I ± 2 ° and either several aliquots of H20 , (Eqn. 2a) or azide (Eqn. 2b) added to obtain BMC I I I or BMC-Az II, respectively. Care was taken to get rid of 02 bubbles before recording the spectrum of BMC n I . Reaction 3 was brought about b y bubbling a stream of CO gas through the solution for I min (avoiding strong light), covering the cuvet, and waiting for 2-3 rain before recording the spectrum. The reactions were carried out at p H 5.4 in order to increase the stability of complexes n and n I (cf. CI-rANCE15). The resulting spectra are shown on Fig. 2. Biochim. Biophys. ,4cta, 54 (1961) 132-144
COMPLEXES OF BACTERIAL CATALASE
z35
0GURA~ had measured the Soret spectra of HBC-Az I I and HBC I I I at p H 7.o and noted that both had an absorption m a x i m u m at the same wavelength, 416 inF. I n our measurements at p H 5.4 it was found that the m a x i m u m of BMC I l l remained 400
I
"~
~,'A
2"
...
A
B ,/o-.
70
......./
60
d
I
/]~
~ 5o ~ 4o
\
',
,
~ ~o ~0
!
Io
EXp! 125
0 |
36o
E=pls A80,
.
~8o
4~0
4oo
4~0
0
4~0
.),(m u}
460
.
.
A70-7AI 9,I3
500
.
.
.
540
.
o...~.-~"
.
.
580
.
.
620
.,V 660
Fig. 2A, the Soret spectra of bacterial catalase (BMC) and some of its derivatives, obtained in t h e Y a n g and Legallais split b e a m recorder. The p H w a s 5.4 (20 m M p h o s p h a t e buffer), the t e m p e r a t u r e as indicated below. The catalase concentration w a s o. 3/~M. Ordinate, extinction coefficient, a s s u m i n g e405 = 405 em-1 mM-X for free BMC 18 ; abscissa, w a v e l e n g t h in m # . A, BMC (free) at 25 °, no addition; B, BMC-MeOOH I I at 25 °, i m M MeOOH; C, BMC-Az I I at I ° as B, plus 5 m M N a N s ; D, BMC-Az I I at I ° as C, plus i m i n CO; E, BMC I I I at I ° as B, plus 5.o. 4 m M H~Oz. Fig. 2]3, The visible region s p e c t r a of the c o m p o u n d s listed in Fig. 2A. The catalase concent r a t i o n was o.6 i~M, all other conditions as described in Fig. 2A. The s p e c t r u m of E is virtually t h e s a m e ~s t h a t of C a n d is therefore o m i t t e d from the figure. 150
,' ,,.-A
I00
I00
50
/
KB
E
0
2t
0 . . . .
ts
'E
-
u
50
~o
'*
I00
°
•~
It~
"~ - J O 0 200 -
Expt. A 128
•
-150
360
400
440
480
%(rap)
Fig. 3- The difference spectra in the Sorer region of some bacterial catalase complexes. BMC concentration, 0. 3 # M , p H 5.4 (20 m M p h o s p h a t e buffer). Ordinate, difference of extinction coefficients in m M -1 cm 1, cf. legend Fig. 2 A; abscissa, w a v e length in m/z. T e m p e r a t u r e , I ° A, difference of the s p e c t r u m of complex I I I (BMC I I I ) m i n u s t h a t of c o m p l e x I I (BMCMeOOH II). B, difference of the s p e c t r u m of the CO c o m p o u n d (BMC-Az II-CO) m i n u s t h a t of BMC I I I .
3ooi 360
380
400
4"20
4i0
4"60
"~, ( r n p )
Fig. 4. The lack of influence of p H on t h e s p e c t r a of complex I I a n d I I I . BMC concent r a t i o n 0. 4/2M. T e m p a r a t u r e N i °. The d r a w n o u t lines represent d a t a obtained at p H 5.4, the full circles those obtained at p H 7.o (20 m M p h o s p h a t e in b o t h cases). The reference solution was always t h a t of free BMC of the s a m e p H as t h a t of t h e c o m p l e x measured. (a) Curve with a m i n i m u m at 4o3 m F : BMC-MeOOH I I m i n u s BMC, b o t h sets of d a t a t a k e n from this paper. (b) Curve w i t h a m i n i m u m a t 439 m/z: BMC I I I m i n u s BMC. The full line from t h i s paper, the full circles a n d d o t t e d line according to OGURA3. A base line error p r o b a b l y a c c o u n t s for m o s t of the difference b e t w e e n these curves.
Biochim. Biophys. Acta, 54 (1961) 132-144
4~ 4~
~o
v
DATA
OF
CATALASES
AND
SOME
OF
THEIR
DERIVATIVES
IN
THE
SORET
REGION
387, + r55 43o.5,--19o
* pH and buffer not stated. ** pH 6.9-7.5, Io-Zo m M phosphate.
Cat-Az II-CO
Cat-Az II
--i28 + 83
--12o +90
37 I, +3o 427.5, - - 5 4
407, 434,
400, 432,
+80 --72
413, 439,
387, + z55 426"5,--z°5
308 / 280** (ref. 36) [ 295 :* (ref. 34) 309 (ref. 40)]
- - * * (ref. 36) - * * (ref. 34)] -(ref. 38)3
Cat-MeOOH I I
428, 429,5, 427, [429,
Cat-H202III
+200 --z55
4zo * (ref. 34) 420 - - * * (ref. 36) 421, 378** (ref. 37)] V419, 1417,
403, 433,
405, 406, F4O5,
I
Cat-MeOOHII
Free catalase
Cat
412, 434,
394, 423,
416 , 4 I6 , [416,
426 426,
413.5 413,
+ z o7 --Ho
--66 +48
364** 334 334(ref. 38)]
- - ( r e f . 38 )
- - (ref. 38)
Cat-Hi02 I I I
37 ° 364** - - * * (ref. 36) 393 * * (ref. 39) 293** (ref. 39)]
- - * * (ref. 36)
4o8, + .r4o 428, - - z z o
412, 416, 417, 418, [416,
414.5
423
412.5 416,
Cat-Az I I
427, 427, [426.5,
421,
420
422.5
4o7
418.5 418,
336 - - * * (ref. 36) - - * (ref. 41) ]
- - * * (ref. 36)
- - * * (ref. 36)
C~-AzlI-CO
Wavelengths (m#) are in bold face, extinction coefficients (cm -1 • m M -t) in italics. The "absolute" peaks are listed inside the lined boxes. The isosbestic points are in the upper right corners. The d a t a in the lower left corners represent the peaks ( + ) a n d troughs (I-) of the difference spectra of the compound listed in the first row minus the compound listed in the first column. The brackets signify [Horse blood catalase] and < Horse liver c a t a l a s e > . No brackets: BMC. pH 5.4, 20 m M phosphate buffer except as noted b y asterisk. No symbol, data from this paper.
SPECTROPHOTOMETRIC
TABLE I
"~
O"
137
COMPLEXES OF BACTERIAL CATALASE
unchanged, while the m a x i m u m of BMC-Az I I had shifted to 412 m/z. These data appear in Table I. The shift of the Soret peak was further demonstrated b y recording the difference spectrum of BMC I I I minus BMC-Az II. The recording is reproduced in Fig. 3, curve A. This shift of the peak is merely an effect of pH, independent of other factors, as shown in an experiment where the reactions I and 2b were carried out at p H 4.2. BMC-Az I I was formed, with a m a x i m u m at 412 m/z. When the p H was now increased to 6. 9 b y the addition of a solution of concentrated secondary phosphate, the peak of BMZ-Az I I shifted to 416 m#. By contrast, the Soret bands T A B L E II SPECTROPHOTOMETRIC
DATA
ON IN
CATALASES
THE
VISIBLE
AND
SOME
OF
THEIR
DERIVATIVES
REGION
F o r e x p l a n a t i o n s see legend of Table I. Catalase
Catalase
Catalase complex MeOOH II
Catalase Az II
see Table I I I
5Ol, 566, 624, 670,
+ -+ --
14 6I 16 I2
550,--3 o 582,--3Z 627, + X9
Cat-MeOOH II
477, 600, 530, 567, [-529, [.565,
Cat-Az II
515, 653
493, 6oo,
50* 38)-] 72 48* (ref. 49 (ref.
548,--2o 567, + 24 587,--z6
38)_11
574,--59 629, + 22
552, _ 2 8 626, + I I
515, 655
490,509, 604,659
5X5' 536 558 , 578 600
607
548 , 582, 548, 584, 548, , 587, 554, 59 o,
5o4,6x6
~559 ~ Catalase Az I I - C O
Cat-Az I1-CO
62 52 68 56 588 (59) (56 ) ---
57 o, - - 33 640, + 7
(ref. (ref. (ref. (ref. (ref. (ref. (ref.
42) 42) 33) 43)\ 43)/ 44)\ I 44)/l Ii I I
573, ~546, \577, 545, 580,
~
81 (57) (54) ---
(ref. (ref. (ref. (ref.
43)\ 43)/ 44)\ 44)//
* p H and buffer n o t stated.
of BMC-MeOOH I I and BMC I I I are apparently very little affected b y changes of p H in the region 5-8 as demonstrated b y the difference spectra plotted in Fig. 4. This finding seems to be at variance with the data compiled b y CHANCEa8 for HBC. In the visible region, the spectral differences between BMC-Az I I and BMC I I I at p H 5-4 and 6. 9 were below the experimental error. When the concentration of catalase was increased, formation of BMC I I I was not complete. The spectral data obtained at p H 5.4 are collected in Table I (Soret region) and Table I I (visible region).
Light dissociation data Since the spectra of BMC-Az I I and BMC I I I in the visible region are virtually the same at p H 5.4 and at p H 6. 9, and their Soret spectra at p H 6. 9 very similar Biochim. Biophys. Acta, 54 (I96t) I 3 2 - I 4 4
138
A.C. MAEHLY
indeed, it was felt that an additional criterium for differentiating between these complexes was desirable. KEILIN AND HARTREE4 had discovered a CO compound of their catalase-H,O,-azide complex and has published the spectrum of this compound in the visible region TM. It was of interest to compare the Soret spectra of BMC-Az I I and BMC-Az II-CO, with a light dissociation spectrum obtained in the double-beam spectrophotometer, using the technique described in METHODS. The high sensitivity of this spectrophotometer made it possible to lower the concentration of the catalase solutions to O.l-O.2 /~M. Appropriate pairs of wavelengths were selected from Table I so as to obtain the maximal deflections (highest peak minus deepest trough in the difference spectra) for studying reactions I to 3. A
ligh~on /light off
L6~~ 8
1.4
Time Expt. A I 4 2
0
I0
20
30
Sec
Fig. 5. A, o. 3 # M B M C - A z I I - C O in 2o m M p h o s p h a t e buffer of p H 6.9 w a s i l l u m i n a t e d (c/. t e x t ) . T h e light dissociation r e a c t i o n a n d t h e following d a r k a s s o c i a t i o n r e a c t i o n were o b s e r v e d in t h e double b e a m s p e c t r o p h o t o m e t e r a t 428-408 m/~. O r d i n a t e , c h a n g e in a b s o r b a n c y difference; abscissa, t i m e ; i division = i m i n . C o n c e n t r a t i o n of CO N 0.9 m M . T e m p e r a t u r e 25 °. B, T h e t w o r e a c t i o n s of Fig. 5 A are r e d r a w n . A l o g a r i t h m i c f u n c t i o n of t h e a b s o r b a n c y is p l o t t e d vs. t i m e .
In this way, the formation of BMC-MeOOH II, BMC-Az II, BMC-Az I I - C O and BMC I I I could be followed as a function of time, in a way similar to that described by OGURA~,3. Once the CO complex had been formed in the dark (Eqn. 3), the projector lamp was turned on and the light dissociation reaction recorded. On reaching the equilibrium the spectrophotometric trace remained stable. When the light was turned off the dark reaction continued unchallenged until dark equilibrium was reached. The reversible dissociation kinetics are shown on the original trace of Fig. 5 A. The kinetic curves were plotted on a semilog scale and are reproduced in Fig. 5 B, showing that both reactions appear to be of the first order. Artifacts in the recorder traces due to the passing of illuminating light into the detector circuit could not be completely eliminated but could easily be corrected for. The velocity of the dark reaction was found to be quite strongly temperature dependent with a Qloo = 3.4 ~ 0.3 at pH 5.4. The pseudo first order velocity constant was computed from the half-time and found to be k'D = (6.5 =L I.O) × lO-2 sec -1 at 25 ° and for o.9"1o -3 M CO (the CO saturation value at 25 ° in pure water is o.93. lO -3 M). The second order velocity constant thus becomes 72 ± 9 M-1 sec-~. The light reaction is almost insensitive to changes of temperature within the range used in these experiments. But the light intensity has, of course, an influence on Biochim. Biophys. Acta, 54 (1961) 132-144
COMPLEXES OF BACTERIAL CATALASE
139
the rate of the dissociation reaction and on the equilibrium. Fig. 6 shows the effect of a 4-fold variation in light intensity (two-fold varidtion in light source distance) on the rate of dissociation. The equilibrium, expressed as the ratio of the concentrations of light dissociated BMC-Az II to dark associated BMC-Az II-CO was found to be proportional to the light intensity and extrapolation to zero intensity gave an equilibrium value of zero. 0,5
0.4
-o
t
0.3
0.2 o 0.1
0 0 I ( I 0 3 Cr'n- 2 )
Fig. 6. The equilibrium of reaction 3 of the t e x t as a function of light intensity. Ordinate, equili[BMC-Az I I ] A d b r i u m , expressed a s t h e ratio I ° ° - I B M C - A z IIJ = i o o - A d where the concentration of thedissociated form is expressed in per cent of the total catalase concentration, i.e. [BMC-Az II1 + EBMCAz II-CO]. Abscissa, relative light i n t e n s i t y I, expressed as the reciprocal s q u a r e of the distance in lO 3 cm -2.
Circumstantial evidence of the presence of a true equilibrium in Eqn. 3 is provided by an experiment where the dissociation of BMC-Az II-CO by light is periodically interrupted by short dark periods, as shown in Fig. 7. The individual traces of the light reaction can be combined to a composite trace that is identical with that of an uninterrupted light reaction. The light dissociation of the CO compound was repeated in a series of experiments at pH 6. 9 (20 m M phosphate) and gave essentially the same kinetic and equilibrium data as at pH 5.4.
4A
--~--
Expt Ill
Composite B
CurVe -
~
T
O.OSO
Fig. 7. A, an original recording obtained b y interm i t t e n t illumination of BMC-Az I I - C O . To a 0.22 # M solution of BMC in 2o m M p h o s p h a t e buffer, p H 5.4, MeOOH was added to a final concentration of o.67 raM. This w a s followed b y an addition of N a N 3 to a final c o n c e n t r a t i o n of 6.7 m M and b y i rain b u b b l i n g w i t h p u r e CO gas. The difference of a b s o r b a n c y at 428 m/~ a n d 4o6 m/t was m e a s u r e d in the double b e a m spectrop h o t o m e t e r as a function of time. Ordinate, change in a b s o r b a n c y (AA); abscissa, time; i u n i t = i min. The time scale r u n s from left to right. The illuminating light b e a m was t u r n e d on and off as indicated on the record. B, the recording of Fig. 7 A is redrawn, b u t only those p a r t s of the reaction where the light was on are included. The resulting composite curve closely resembles a typical, u n i n t e r r u p t e d light reaction trace such as t h a t of Fig. 5 A.
Biochim. Biophys. Acta, 54 (1961) 132-144
140
A. C. MAEHLY
I t remained to be seen if the light dissociation of BMC-Az I I - C O really leads to BMC-Az II, or if another ~omplex is formed in this reaction. To this end, a light dissociation spectrum was taken b y performing the dissociation and association reactions at a series of wavelengths through the region of the Soret spectrum. As reference wavelength 412 m F was chosen, close to the peak of the expected difference spectrum between the two complexes involved. Since, at the equilibrium in the light, the CO complex is only partially dissociated, partial deflections were recorded. The values thus obtained were compared with the difference spectrum of BMC-Az I I - C O minus BMC-Az I I (recorded on the Yang-Legallais spectrophotometer) b y multiplying them by an arbitrary constant factor. The data in Fig. 8 show that the light dissociation does indeed lead to the formation of BMC-Az II. If intermediates are formed, their lifetime is below the time resolution of the method used here ( ~ I sec). Fig. 8. The difference s p e c t r u m of BMCAz I I - C O m i n u s BMC-Az I I , o b t a i n e d in t w o different ways. I. The u n b r o k e n trace is an original recording (Expt. A 127) o b t a i n e d in the Y a n g - L e g a l l a i s a p p a r a t u s . Conditions: BMC c o n c e n t r a t i o n o . 4 4 / z M in 2o m M p h o s p h a t e buffer, p H 5.4. BMC-Az I I was formed b y the addition of 0.67 m M MeOOH and 6.7 m M azide. Two c u v e t s were filled w i t h the resulting solution. P u r e CO w a s b u b b l e d t h r o u g h one of the c u r e t s for I rain, the c u r e t closed w i t h a greased cover, a n d I i P , r i I ~ P , I , the difference s p e c t r u m recorded in the d a r k 32O 340 360 380 400 420 440 460 480 500 a n d at 25 ° . 2. The open circles represent individual readings in light dissociation exp e r i m e n t s of the t y p e represented in Fig. 5 A. Conditions of the e x p e r i m e n t (A 116) : BMC concent r a t i o n o.16/~M in 20 m M p h o s p h a t e buffer, p H 5.4. The addition of 0.9 m M MeOOH and 6.5 m M azide was followed b y i n t e r m i t t e n t b u b b l i n g of p u r e CO for I-rain periods to form BMC-Az I I - C O . The distance f r o m the illuminating light source was 16 cm. One w a v e l e n g t h of t h e double, b e a m i n s t r u m e n t of CHANCE was set at 412 m/~, for the o t h e r w a v e l e n g t h various values b e t w e e n 360 a n d 460 m # were chosen. At each selected pair of w a v e l e n g t h s the dark a n d / o r light equilibrium values of the a b s o r b a n c y difference were recorded. The whole set of d a t a t h u s o b t a i n e d was plotted in the figure, using an empirical factor for bringing the d a t a to the same scale as t h a t of the recorded difference s p e c t r u m .
Since the experimental conditions for the formation of the CO compound from BMC-Az I I had been clearly established, efforts were now made to demonstrate a CO binding of complex III, both at p H 5.4 and p H 6. 9. In some experiments, complex I I I was formed in the usual way (several additions of small concentrations of H202, Eqn. 2a) and CO bubbled through the solution; in others, the solution of complex I I was saturated with CO and H20 ~ added afterwards. In neither case was there any spectral change observed, either in the dark or during illumination. Working with an anaerobic cuvet 17 in an atmosphere of pure CO, and adding H~O~ from a sidearm gave the same results. In order to see if H~O 2 itself or one of its reaction products (02!) acted as an inhibitor, two kinds of tests were carried out: (a) the formation of BMC-Az I I - C O was allowed to go to completion (dark equilibrium), then successive additions of H~O~ were m a d e - - m o r e than would be needed to form complex I I I from complex II. No spectral change was observed. (b) Conversely, complex I I I was formed in a CO atmosphere. Formation of a CO complex could Biochim. Biophys. Acta, 54 (1961) 132-144
4~
?
~o
~D
el/H
971x, o 4 . 9.5
405 405, 405,
Io2" * * 4o5, x o 2 * * * 406, z o 5 * * *
BMC BMC BMC BMC
--
--
e2/H
5Ol, 9.o*** 505, x z . 4
9.9 ~*~
9.2***
--
--
--
541 , 8 . 6 " * * 538, z o . 2 * * *
545,
5 o6, x o . 7 * * *
545
(536)
536
. 541 ,
--
--
e3/H
536, XT*
54 °
23
. 5o5, t o . 5 ~*~
5°6
503, x 7 . 6 " *
502
505
.~
BANDS
OF
CATALASES
.
edH
63o, 629,
631,
623,
7.z*** 7.2
8.4***
. 7-3***
631 - 626, ( ~ 6)
627, x 2 . 3 " *
624, x o . 6
627
622 -622, x o . 8 623, .rs~ 623, 9
?q
-12.5 14. 4 14.6
13.°
-----
-~2.9 9.5 --__ -13.o
ex e~t
(~) in m/~ ; e / H , e x t i n c t i o n c o e f f i c i e n t p e r h e m i n , in c m -1 × m M - L
ABSORPTION
III
2. 4 H e r o e s . ** 4 H e m e s . * ** C a l c u l a t e d f r o m AGNER'S v a l u e s a n d a s s u m i n g t h a t h e h a d d i v i d e d t h e s e b y 4-
4o5,
95***
405,
405, x 3 8 " 405, x 3 8
405, I 4 2 "
409, x 4 5 4o0, x 4 2 "
2x
HBC HBC HBC HBC HBC HBC
HLC HLC HLC HLC HLC HLC HLC HLC
Type of catalase
Wavelength
TABLE
6.8 -5.4-8.2 - - 7 .2
6.8
7.1 7.I 6.8 --
? ? 7.x 7.1 ---6.0
pH
33 34 This paper 35
29 27 3° 31 32 This paper
2~ 22 23 24 25 26 27 28
Reference
i-4
oq
,.4 t'rl
o~ ©
,.~
©
('3
142
A. C. MAEHLY
not be observed. After addition of 2.5 m M azide, BMC-Az I I - C O was fully formed (dark-equilibrium). These experiments show that only BMC-Az I I but not complex I I I is able to bind carbon monoxide. DISCUSSION
Since both the Soret spectra and the spectra in the visible region were to be compared, a survey of the literature was made in order to find spectral differences in the three most intensively studied types of catalase: horse blood catalase, horse liver catalase, and bacterial micrococcus catalase. In spite of the thousands of publications on these enzymes, very few quantitative spectrophotometric data were found. This is partly due to the lack of adequate spectrophotometers prior to the second World War, partly to the fact that most research was done either in the visible or in the Soret region, but only rarely in both. HERBERT AND PINSENTe calculated their spectral data in the visible range from dry weight determinations of their crystalline bacterial catalase. They found ee31 = 32.4 cm -1 m M -1 (from E % ----- 1.47 ), but gave no values for the Soret region. - - i~cm CHANCE AND HERBERT18 found the extinction coefficient at 405 m ~ to be 405 i 5 cm-1 mM-1, this time based on the measurement of the hemin content of BMC b y forming the pyridin-hemochromogen. The ratio of these two values is ea0Jee31 = 12.5. A direct comparison was made by Dr. A. BRILL b y diluting a stock solution of BMC appropriately with phosphate buffers of p H 5.9-8.2. A ratio eao5/e630 = 14.6 ± 0.2 was obtained. The pyridin hemochromogen method, which was considered more reliable than the dry weight determination, gave a value of e405 = 405 cm -1 m M -1., the same figure as CHANCE AND HERBERT18. I t was taken as the primary standard, and eeso becomes thus 405 :[: 5/14.6 = 28.7 ± 0.3 cm -1 m M -1. The ratio e4oJee30 is higher for BMC than for HBC or HLC. Table I I I gives a review of the published spectral data for the three catalases. It is surprising to see how incomplete these spectral data are in spite of the extent of the literature on catalases. Table I lists the position and intensity of the Soret bands both for the absolute spectra and difference spectra of BMC, BMC-MeOOH II, BMC-H~O, III, BMC-Az I I and BMC-Az II-CO. Some values for HBC and HLC are included. The table proved useful in the choice of appropriate wave lengths for the rate and equilibrium studies with the double beam spectrophotometer. The spectral data for the visible region are compiled in Table I I in the same manner as in Table I. Here a considerable discrepancy is found between the absolute spectra of BMC-Az I I (and its CO complex) and HLC-Az I I (and its CO complex). The spectrum published by KEILIN AND HARTREEle for HLC-Az I I - C O in particular has a very different shape from that observed here for BMC-Az II-CO. This is made clearer by a glance at Fig. 9 which gives the difference spectra of these compounds minus free catalase. Plotted in this way, comparison between HLC and BMC is facilitated. The light dissociation data presented here certainly confirm KEILIN AND HARTREE'S observations, and extend them to include kinetic measurements. However, * I n t h e p u r e s t p r e p a r a t i o n s of BMC, a v a l u e of ~o5 = 420 c m-1 m M - 1 , close t o t h e h i g e s t v a l u e p u b l i s h e d b y DEUTSCH 19 :[or HBC, a n o t h e r 4-heroin c a t a l a s e , w a s o b t a i n e d b y Dr. A.BRILL 2°.
Biochim. Biophys. Acta, 54 (1961) 132-144
143
C O M P L E X E S OF B A C T E R I A L CATALASE
experimental difficulties have so far prevented a calculation of the actual rate and equilibrium constants. These difficulties stem from the competition of 0 , and CO for Cat-Az II. Working under the usual conditions of anaerobiosis, i.e., in an inert gas, does not remove all oxygen from Cat-Az II. Addition of sodium dithionite does not affect the CO compound but decomposes Cat-Az II rapidly. The only experiments where the CO concentration was known were those at complete C0-saturation, achieved by extensive gassing of the solution. Further experimentation will be needed to secure the C0-titration data necessary for computing the true constants for the dark and light reactions. The fact that no CO binding could be observed with catalase complex III under the conditions of the experiments reported here has the obvious limitations of all negative evidence. Until such a CO binding can be demonstrated, there is no reason to postulate Complex III as a ferrous compound. // 50
/ ,,
',,
/
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F i g . 9- D i f f e r e n c e s p e c t r a of s o m e c a t a l a s e derivatives in the visible region. The differences between the s p e c t r a of B M C - A z I [ a n d B M C - A z I I - C O , r e s p e c t i v e l y and the s p e c t r u m of free B M C a r e plotted. F o r c o m p a r i s o n , t h e s p e c t r a of H L C - A z I I a n d H L C - A z I I - C O r e s p e c t i v e l y versus the s p e c t r u m of H L C a r e p l o t t e d f r o m t h e p a p e r of KEILIN AND HARTREE 16. C, - - - - , B M C - A z I I vs. BMC. C a t a l a s e concentration in b o t h c u v e t s , 0 . 6 / z M . T e m p e r a t u r e + 2 °. 20 m M p h o s p h a t e b u f f e r of p H 5.4. A d d i t i o n s : 2 m M M e O O H + IO m M N a N 3 t o free BMC. D, - . . . . , B M C A z I I - C O vs. BMC. T h e s o l u t i o n w a s o b t a i n e d b y bubbling CO for I min through solution C. I, X - - X , H L C - A z IT vs. H L C , r e p l o t t e d f r o m Fig. I i n KEILIN AND HARTREE'S p a p e r 16. C a t a l a s e concentration not given, p H 6.8. N 2 a t m o s p h e r e . 2, C) ..... © , H L C A z I I - C O vs. H L C , plotted a s for c u r v e I.
ACKNOWLEDGEMENTS The author wants to thank Dr. B. CHANCE for his interest and encouragement and Dr. A. BRILL for detai]ed discussions. Thanks are due to Mr. L. PAKMAN and Mr. M. BARR for their cooperation in preparing the enzyme. The work was supported in part by a grant from the U.S. Public Health Service, Division of Grants and Fellowships. REFERENCES 1 y . OGURA, H. HASEGAWA, K. NAGANO AND K. OGASAWARA, Symposia on E n z y m e Chem., Japan, 9 (1954) 91. 2 y . OGURA, Arch. Biochem. Biophys., 57 (1955) 288. 3 y . OGURA, p e r s o n a l communication. 4 D. KEILIN AND E . F. HARTREE, Proc. Roy. Soc. (London) B, 121 (1936) 173. Biochim. Biophys. Acta, 54 (1961) 1 3 2 - 1 4 4
~44
A.C. M A E H L Y
5 D. KEILIN AND E. F. HARTREE, Biochem. J., 49 (1951) 88. 6 D. HERBERT AND J. PINSENT, Biochem J., 43 (1948) 203. 7 R. F. BEERS, Science, 122 (1955) lO16. 8 A. BRILL AND A. C. MAEHLY, to be published. 9 D. HERBERT AND J. PINSENT, Biochem. J., 43 (1948) 193. 10 C. C. YANG AND V. LEGALLAIS, Rev. Sci. Instr., 25 (1955) 8Ol. 11 C. C. YANG, Rev. Sci. Instr., 25 (1955) 807. 12 B. CHANCE, Rev. Sci. Instr., 22 (1951) 634. 13 B. CHANCE, L. SMITH AND L. CASTOR, Biochim. Biophys. Acta, 12 (1953) 289. 14 K. AGNER AND H. THEORELL, Arch. Biochem., io (1946) 321. 15 B. CHANCE, J. Biol. Chem., 194 (1952) 471. 16 D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 148. 17 A. C. MAEHLY, L. SMITH AND J. D. GRAHAM, Jr., Science, 122 (1955) 767. 18 B. CHANCE AND D. HERBERT, Biochem. J., 46 (195o) 402. 16 H. F. DEUTSCH, Aeta Chem. Scan&, 6 (1952) 1516. 2o A. BRILL, personal c o m m u n i c a t i o n . 21 K. G. STERN, J. Gen. Physiol., 20 (1937) 631. 22 K. G. STERN, J. Biol. Chem., 121 (1937) 561. 23 K. AGNER, Biochem. J., 32 (1938) 17o2. 24 K. AGNER, Arkiv. Kemi Mineral. Geol., 16A (1942) No. 6. 25 A. L. DOUNCE AND O. D. FRAMPTON, Science, 89 (1939) 300. 26 j . B. SUMNER, Advances in Enzymology, Vol. i, Interscience Publishers, 1941, p. 163. 27 R. K. BONNICHSEN, Arch. Biochem., 12 (1947) 88. 26 D. KEILIN AND E. V. HARTREE, Biochem. J., 49 (1951) 88. 26 K. AGNER, Arhiv. Kemi Mineral. Geol., I7B (1943) No. 9. 30 D. HERBERT AND J. PINSENT, Biochem. J., 43 (1948) 203. 31 H. F. DEUTSCH, Acta Chem. Scan&, .5 (1951) lO74. 32 H. F. DEUTSCH, Acta Chem. Scand., 6 (1952) 1516. 33 D. HERBERT AND J. PINSENT, Biochem. J., 43 (1948) 193. 34 B. CHANCE AND D. HERBERT, Biochem. J., 46 (195 o) 402. 35 A. BRILL, personal c o m m u n i c a t i o n . 36 y . OGURA AND A. EHRENBERG, results obtained at + 9 ° in a B e c k m a n D U s p e c t r o p h o t o m e t e r a t the Nobel Medical I n s t i t u t e , Stockholm. 37 R. K. BONNICHSEN, Arch. Biochem., 12 (1947) 83. 36 B. CHANCE, Arch. Biochem. Biophys., 41 (1952) 404 . 39 y . OGURA, personal c o m m u n i c a t i o n . 40 H. THEORELL AND A. EHRENBERG, Arch. Biochem. Biophys., 41 (1952) 442. 41 H. THEORELL AND A. EHRENBERG, personal communication. 42 y . OGURA, personal c o m m u n i c a t i o n . 43 D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 148. The e values per h e m i n were multiplied b y 4. 44. H. THEORELL AND K. AGNER, Arkiv Kemi, Mineral. Geol., I6A (1942) No. 7.
Bioehim. Biophys. Acta, 54 (1961) 132-144
A. MUHAMMED
5 A. KORNBERG, S. R. I~ORNBERG AND E. S. SIMMS, Biochim. Biophys. Acta, 20 (1956) 215. 6 S. R. KORNBERG, Biochim. Biophys. Acta, 26 (1957) 294. F. G. WINDER AND J. M. DENNEN¥, Nature, 175 (1955) 636. 8 F. G. WINDER AND J. M. DENNENY, J. Gen. Microbiol., 17 (1957) 573. 9 0 . HOFFMANN-OSTENHOF AND L. SLECHTA, Proc. 2nd. Intern. Syrup. on Enzyme Chem., Japan, (1957) 18o. 10 A. MUHAMMED, A. RODGERS AND D. E. MUGHES, J. Gen. Microbiol., 20 (1959) 482. 11 D. E. MOGHES, Brit. J. Exptl. Pathol., 32 (1951) 97. 12 I. A. ROSE, M. GRUNBERG-MANAGO,S. R. KOREY AND S. OCHOA,J. Biol. Chem., 211 (1954) 737. 13 A. W. D. AVlSON, J. Chem. Soc., (1955) 732. 14 F. LIPPMAN AND L. C. TUTTLE, J.Biol. Chem., 159 (1945) 21. 15 I. BERENBLUM AND E. B. CHAIN, Biochem. J., 32 (1938) 295. is H. A. KREBS AND R. HEMS, Biochim. Biophys. Acta, 12 (1953) 172. 17 G. G. BERG, Anal. Chem., 3 ° (1958) 213. 18 C. S. HANES AND F. A. ISHERWOOD, Nature, 164 (1949) 11o7. 19 W. J. NICKERSON, Experientia, 5 (1949) 202. 2o E. BUKHOVlCH AND A. N. BELOZERSK¥, J. Biochem. (U.S.S.R.), 23 (1958) 238. 31 S. SPIEGELMAN, in W. D. McELROY AND ]3. GLASS, The Chemical Basis o[ Heredity, The J o h n s H o p k i n s Press, p. 232. 22 ~¢1. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Biochim. Biophys. Acta, 20 (1956) 269. 23 S. E. BRESSLER AND KH. M. RUBINA, Ann. Rev. Biochem., (1955) 628. 24 A. A. HAKIM, Enzymologia, 19 26 (1958) 96.
Biochim. Biophys. Acta, 54 (1961) 121-132
COMPLEXES OF BACTERIAL CATALASE WITH PEROXIDE,
AZIDE
AND CARBON MONOXIDE A. C. M A E H L Y
E. R. Johnson Foundation/or Medical Physics, University o/Pennsylvania, Philadelphia, Pa. (U.S.A.) and Government Laboratory/or Forensic Chemistry, Stockholm (Sweden) * (Received April I7th, 1961)
SUMMARY
Using crystalline bacterial catalase, the spectra of peroxide- and peroxide-azide complexes were recorded at pH 5-4 and 7.0. The kinetics of the formation of the carbon monoxide compound of one of these, and its reversible light dissociation was measured. By repeating this reversible process at a series of wavelengths, a light dissociation spectrum was obtained which proved to be identical with the difference spectrum between Cat-Az II and Cat-Az II-CO. Catalase-peroxide complex III has a spectrum very similiar to that of Cat-Az II at pH 7.0. However, by lowering the pH to 5.4, the peak of complex III is unchanged whereas the peak of Cat-Az II moves from 416 m/~ to 412 m/z. Complex III did not bind CO under the conditions of these experiments. The data indicate that Cat-Az II and complex III are not identical. Quantitative spectrophotometric data on catalase from liver, blood and bacteria, as well as on the complexes mentioned above, are listed. They are compared to earlier data in the literature. N o n - s t a n d a r d a b b r e v i a t i o n s : HLC, horse liver catalase; HBC, horse blood catalase; BMC, bacterial micrococcus catalase. * Mailing address: G o v e r n m e n t L a b o r a t o r y for Forensic Chemistry, Tomtebodaviigen 3o, Stockholm 60, Sweden.
Biochim. Biophys. Acta, 54 (1961) 132-144
COMPLEXES OF BACTERIAL CATALASE
133
INTRODUCTION
OGURA et al. 1-s have shown that catalase with a hydro-peroxide (H,O2, CH3OOH, C,HsOH ) and azide forms two distinct complexes which he calls Cat-Az I and Cat-Az II. The latter was shown to be identical with the catalase-azide-H,O 2 complex first described b y KEIL1N AND HARTREE4. The spectrum of Cat-Az I I in the visible region was found to be so closely similar to that of c a t a l a s e - H 2 0 , - c o m plex I I I , also discovered b y KEILIN AND HARTREE s, that the question arises if these two complexes are identical as well. The present paper reports spectrophotometric and light dissociation data which, it is hoped, will help to clear up this question. I t is well known that HLC contains varying amounts of bile pigment and that HBC is hard to rid of traces of hemoglobin. BMC seems to have neither of these disadvantages: I t has the full complement of 4 hemingroups (like HBC), and has not been shown to be accompanied b y heroin impurities. For this reason, BMC was selected for the present studies. EXPERIMENTAL
The HBC used for the spectrophotometric data presented in Tables I - I I I was prepared in collaboration with Dr. W. J. STEELE according to the method of HERBERT A N D PINSENTs. After several recrystallizations, a RZ ( A i o s / A 28o) of 1.21 was obtained. The Kat.f. was 60000. Micrococcus L was grown according to BEERS7. and the enzyme (BMC) prepared and crystallized b y a modification s of the method of HERBERT AND PINSENT9. The preparation employed here had a RZ of 0.78. Two different recording spectrophotometers were used in this work. Spectra of all compounds described were recorded on a chopped beam spectrophotometer devised b y YANG AND LEGALLAIS10,11, either at low speed (approx. 30 m/,/min) or, if the compounds were unstable, at high speeds of about 2-6 mF/sec. "Absolute" spectra (enzyme vs. buffer) as well as "difference" spectra (enzyme vs. enzyme + reactant) were recorded. A double beam instrument designed by CHANCETM was used for studying 100 5543
~ 2o ~
o
350
400
450
(m.u)
500
550
.
600
Fig. i. The t r a n s m i s s i o n of the filters used for the m e a s u r e m e n t of the kinetics of the light dissociation. Shaded areas: light. The illuminating light is cut off below 55o m/~, the p h o t o m u l t i p l i e r receives light only in the Soret region. Abscissa, w a v e length in m/~; ordinate, t r a n s m i s s o n in percent. The curves are replotted from the suppliers catalogue (Corning Glass Works, Coming, New Hampshire). * The a u t h o r is v e r y grateful to Dr. R. F. BEERS, Jr. for detailed instructions on the t e c h n i q u e s used for growing the organism.
Biochim. Biophys. Acta, 54 (1961) 132-144
134
A.C. MAEHLY
the formation and the reversible dissociation by light of the carbon monoxide complex of Cat-Az n . This apparatus measures the difference of absorbancy at two freely chosen wave lengths as a function of time. For the study of the light dissociation, light from a Ioo-W projector bulb operated on direct current was allowed to fall on the cuvet at right angles to that of the measuring beams. The absorbancies were recorded with the help of the amplified alternating current output of a photomultiplier tube. The technique employed is essentially that of CHANCE, SMITH AND CASTOR13, except that the photocell was replaced b y a head-on type photomultiplier tube. A Coming Filter No. 3482 w a s used at the entrance opening (illuminating light), a No. 5543 filter was inserted between sample and photomultiplier. The filter arrangement is shown on Fig. i. In both instruments, cooling was provided b y an alcoholwater b a t h at - - i o ° and a pump. The temperature in the I-cm cuvets could thus be kept a + I ± 2 °. For forming CO compounds, CO gas from a tank was passed through the solutions and the cuvet closed with a greased cover immediately afterwards. RESULTS
Absorption spectra In order to obtain the absorption spectra of free catalase (BMC), of MeOOH complex I I {BMC-MeOOH II), of H20 2 complex I I I (BMC I I I ) of peroxide-azide complex I I (BMC-Az II), and finally, of the CO compound (BMC-Az II-CO) found b y KEILIN AND HARTREE 4, the chopped beam recorder of YANG AND LEGALLAIS w a s used. In a typical experiment, the sequence of reactions and the concentrations of the reactants were the following: BMC + MeOOH (0. 3/~M) (I mM)
\
BMC-MeOOH II + H,O, (0. 3/zM)
(5.0.4 mM)
BMC-MeOOH II + NaN s (0.3/~M) (5 mM)
BMC-Az II + CO (o-3/tM)
25°\
BMC-MeOOH II
(I)
~ I°\
BMC III
(2a)
~ I°\
BMC-Az II
(2b)
~ I°\
BMC-Az II-CO
\ \
(3)
2o m M phosphate buffer was used throughout, since the work of AGNER AND THEORELL14 and of CHANCE15 had shown that catalase binds m a n y ions strongly but phosphate ions only to a minor degree (pK'Fe, I-I,PO,, ---- 1.5, (see ref. 14). The concentration of catalase never exceeded 0.5 ~ M and was usually kept lower, since it was found that the higher the concentration of catalase, the more H20 , was needed to shift the equilibrium of reaction (2a) to the right. Complex I I was formed at room temperature in order to let the reaction go to completion within reasonable time. The solution was then cooled to + I ± 2 ° and either several aliquots of H20 , (Eqn. 2a) or azide (Eqn. 2b) added to obtain BMC I I I or BMC-Az II, respectively. Care was taken to get rid of 02 bubbles before recording the spectrum of BMC n I . Reaction 3 was brought about b y bubbling a stream of CO gas through the solution for I min (avoiding strong light), covering the cuvet, and waiting for 2-3 rain before recording the spectrum. The reactions were carried out at p H 5.4 in order to increase the stability of complexes n and n I (cf. CI-rANCE15). The resulting spectra are shown on Fig. 2. Biochim. Biophys. ,4cta, 54 (1961) 132-144
COMPLEXES OF BACTERIAL CATALASE
z35
0GURA~ had measured the Soret spectra of HBC-Az I I and HBC I I I at p H 7.o and noted that both had an absorption m a x i m u m at the same wavelength, 416 inF. I n our measurements at p H 5.4 it was found that the m a x i m u m of BMC I l l remained 400
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620
.,V 660
Fig. 2A, the Soret spectra of bacterial catalase (BMC) and some of its derivatives, obtained in t h e Y a n g and Legallais split b e a m recorder. The p H w a s 5.4 (20 m M p h o s p h a t e buffer), the t e m p e r a t u r e as indicated below. The catalase concentration w a s o. 3/~M. Ordinate, extinction coefficient, a s s u m i n g e405 = 405 em-1 mM-X for free BMC 18 ; abscissa, w a v e l e n g t h in m # . A, BMC (free) at 25 °, no addition; B, BMC-MeOOH I I at 25 °, i m M MeOOH; C, BMC-Az I I at I ° as B, plus 5 m M N a N s ; D, BMC-Az I I at I ° as C, plus i m i n CO; E, BMC I I I at I ° as B, plus 5.o. 4 m M H~Oz. Fig. 2]3, The visible region s p e c t r a of the c o m p o u n d s listed in Fig. 2A. The catalase concent r a t i o n was o.6 i~M, all other conditions as described in Fig. 2A. The s p e c t r u m of E is virtually t h e s a m e ~s t h a t of C a n d is therefore o m i t t e d from the figure. 150
,' ,,.-A
I00
I00
50
/
KB
E
0
2t
0 . . . .
ts
'E
-
u
50
~o
'*
I00
°
•~
It~
"~ - J O 0 200 -
Expt. A 128
•
-150
360
400
440
480
%(rap)
Fig. 3- The difference spectra in the Sorer region of some bacterial catalase complexes. BMC concentration, 0. 3 # M , p H 5.4 (20 m M p h o s p h a t e buffer). Ordinate, difference of extinction coefficients in m M -1 cm 1, cf. legend Fig. 2 A; abscissa, w a v e length in m/z. T e m p e r a t u r e , I ° A, difference of the s p e c t r u m of complex I I I (BMC I I I ) m i n u s t h a t of c o m p l e x I I (BMCMeOOH II). B, difference of the s p e c t r u m of the CO c o m p o u n d (BMC-Az II-CO) m i n u s t h a t of BMC I I I .
3ooi 360
380
400
4"20
4i0
4"60
"~, ( r n p )
Fig. 4. The lack of influence of p H on t h e s p e c t r a of complex I I a n d I I I . BMC concent r a t i o n 0. 4/2M. T e m p a r a t u r e N i °. The d r a w n o u t lines represent d a t a obtained at p H 5.4, the full circles those obtained at p H 7.o (20 m M p h o s p h a t e in b o t h cases). The reference solution was always t h a t of free BMC of the s a m e p H as t h a t of t h e c o m p l e x measured. (a) Curve with a m i n i m u m at 4o3 m F : BMC-MeOOH I I m i n u s BMC, b o t h sets of d a t a t a k e n from this paper. (b) Curve w i t h a m i n i m u m a t 439 m/z: BMC I I I m i n u s BMC. The full line from t h i s paper, the full circles a n d d o t t e d line according to OGURA3. A base line error p r o b a b l y a c c o u n t s for m o s t of the difference b e t w e e n these curves.
Biochim. Biophys. Acta, 54 (1961) 132-144
4~ 4~
~o
v
DATA
OF
CATALASES
AND
SOME
OF
THEIR
DERIVATIVES
IN
THE
SORET
REGION
387, + r55 43o.5,--19o
* pH and buffer not stated. ** pH 6.9-7.5, Io-Zo m M phosphate.
Cat-Az II-CO
Cat-Az II
--i28 + 83
--12o +90
37 I, +3o 427.5, - - 5 4
407, 434,
400, 432,
+80 --72
413, 439,
387, + z55 426"5,--z°5
308 / 280** (ref. 36) [ 295 :* (ref. 34) 309 (ref. 40)]
- - * * (ref. 36) - * * (ref. 34)] -(ref. 38)3
Cat-MeOOH I I
428, 429,5, 427, [429,
Cat-H202III
+200 --z55
4zo * (ref. 34) 420 - - * * (ref. 36) 421, 378** (ref. 37)] V419, 1417,
403, 433,
405, 406, F4O5,
I
Cat-MeOOHII
Free catalase
Cat
412, 434,
394, 423,
416 , 4 I6 , [416,
426 426,
413.5 413,
+ z o7 --Ho
--66 +48
364** 334 334(ref. 38)]
- - ( r e f . 38 )
- - (ref. 38)
Cat-Hi02 I I I
37 ° 364** - - * * (ref. 36) 393 * * (ref. 39) 293** (ref. 39)]
- - * * (ref. 36)
4o8, + .r4o 428, - - z z o
412, 416, 417, 418, [416,
414.5
423
412.5 416,
Cat-Az I I
427, 427, [426.5,
421,
420
422.5
4o7
418.5 418,
336 - - * * (ref. 36) - - * (ref. 41) ]
- - * * (ref. 36)
- - * * (ref. 36)
C~-AzlI-CO
Wavelengths (m#) are in bold face, extinction coefficients (cm -1 • m M -t) in italics. The "absolute" peaks are listed inside the lined boxes. The isosbestic points are in the upper right corners. The d a t a in the lower left corners represent the peaks ( + ) a n d troughs (I-) of the difference spectra of the compound listed in the first row minus the compound listed in the first column. The brackets signify [Horse blood catalase] and < Horse liver c a t a l a s e > . No brackets: BMC. pH 5.4, 20 m M phosphate buffer except as noted b y asterisk. No symbol, data from this paper.
SPECTROPHOTOMETRIC
TABLE I
"~
O"
137
COMPLEXES OF BACTERIAL CATALASE
unchanged, while the m a x i m u m of BMC-Az I I had shifted to 412 m/z. These data appear in Table I. The shift of the Soret peak was further demonstrated b y recording the difference spectrum of BMC I I I minus BMC-Az II. The recording is reproduced in Fig. 3, curve A. This shift of the peak is merely an effect of pH, independent of other factors, as shown in an experiment where the reactions I and 2b were carried out at p H 4.2. BMC-Az I I was formed, with a m a x i m u m at 412 m/z. When the p H was now increased to 6. 9 b y the addition of a solution of concentrated secondary phosphate, the peak of BMZ-Az I I shifted to 416 m#. By contrast, the Soret bands T A B L E II SPECTROPHOTOMETRIC
DATA
ON IN
CATALASES
THE
VISIBLE
AND
SOME
OF
THEIR
DERIVATIVES
REGION
F o r e x p l a n a t i o n s see legend of Table I. Catalase
Catalase
Catalase complex MeOOH II
Catalase Az II
see Table I I I
5Ol, 566, 624, 670,
+ -+ --
14 6I 16 I2
550,--3 o 582,--3Z 627, + X9
Cat-MeOOH II
477, 600, 530, 567, [-529, [.565,
Cat-Az II
515, 653
493, 6oo,
50* 38)-] 72 48* (ref. 49 (ref.
548,--2o 567, + 24 587,--z6
38)_11
574,--59 629, + 22
552, _ 2 8 626, + I I
515, 655
490,509, 604,659
5X5' 536 558 , 578 600
607
548 , 582, 548, 584, 548, , 587, 554, 59 o,
5o4,6x6
~559 ~ Catalase Az I I - C O
Cat-Az I1-CO
62 52 68 56 588 (59) (56 ) ---
57 o, - - 33 640, + 7
(ref. (ref. (ref. (ref. (ref. (ref. (ref.
42) 42) 33) 43)\ 43)/ 44)\ I 44)/l Ii I I
573, ~546, \577, 545, 580,
~
81 (57) (54) ---
(ref. (ref. (ref. (ref.
43)\ 43)/ 44)\ 44)//
* p H and buffer n o t stated.
of BMC-MeOOH I I and BMC I I I are apparently very little affected b y changes of p H in the region 5-8 as demonstrated b y the difference spectra plotted in Fig. 4. This finding seems to be at variance with the data compiled b y CHANCEa8 for HBC. In the visible region, the spectral differences between BMC-Az I I and BMC I I I at p H 5-4 and 6. 9 were below the experimental error. When the concentration of catalase was increased, formation of BMC I I I was not complete. The spectral data obtained at p H 5.4 are collected in Table I (Soret region) and Table I I (visible region).
Light dissociation data Since the spectra of BMC-Az I I and BMC I I I in the visible region are virtually the same at p H 5.4 and at p H 6. 9, and their Soret spectra at p H 6. 9 very similar Biochim. Biophys. Acta, 54 (I96t) I 3 2 - I 4 4
138
A.C. MAEHLY
indeed, it was felt that an additional criterium for differentiating between these complexes was desirable. KEILIN AND HARTREE4 had discovered a CO compound of their catalase-H,O,-azide complex and has published the spectrum of this compound in the visible region TM. It was of interest to compare the Soret spectra of BMC-Az I I and BMC-Az II-CO, with a light dissociation spectrum obtained in the double-beam spectrophotometer, using the technique described in METHODS. The high sensitivity of this spectrophotometer made it possible to lower the concentration of the catalase solutions to O.l-O.2 /~M. Appropriate pairs of wavelengths were selected from Table I so as to obtain the maximal deflections (highest peak minus deepest trough in the difference spectra) for studying reactions I to 3. A
ligh~on /light off
L6~~ 8
1.4
Time Expt. A I 4 2
0
I0
20
30
Sec
Fig. 5. A, o. 3 # M B M C - A z I I - C O in 2o m M p h o s p h a t e buffer of p H 6.9 w a s i l l u m i n a t e d (c/. t e x t ) . T h e light dissociation r e a c t i o n a n d t h e following d a r k a s s o c i a t i o n r e a c t i o n were o b s e r v e d in t h e double b e a m s p e c t r o p h o t o m e t e r a t 428-408 m/~. O r d i n a t e , c h a n g e in a b s o r b a n c y difference; abscissa, t i m e ; i division = i m i n . C o n c e n t r a t i o n of CO N 0.9 m M . T e m p e r a t u r e 25 °. B, T h e t w o r e a c t i o n s of Fig. 5 A are r e d r a w n . A l o g a r i t h m i c f u n c t i o n of t h e a b s o r b a n c y is p l o t t e d vs. t i m e .
In this way, the formation of BMC-MeOOH II, BMC-Az II, BMC-Az I I - C O and BMC I I I could be followed as a function of time, in a way similar to that described by OGURA~,3. Once the CO complex had been formed in the dark (Eqn. 3), the projector lamp was turned on and the light dissociation reaction recorded. On reaching the equilibrium the spectrophotometric trace remained stable. When the light was turned off the dark reaction continued unchallenged until dark equilibrium was reached. The reversible dissociation kinetics are shown on the original trace of Fig. 5 A. The kinetic curves were plotted on a semilog scale and are reproduced in Fig. 5 B, showing that both reactions appear to be of the first order. Artifacts in the recorder traces due to the passing of illuminating light into the detector circuit could not be completely eliminated but could easily be corrected for. The velocity of the dark reaction was found to be quite strongly temperature dependent with a Qloo = 3.4 ~ 0.3 at pH 5.4. The pseudo first order velocity constant was computed from the half-time and found to be k'D = (6.5 =L I.O) × lO-2 sec -1 at 25 ° and for o.9"1o -3 M CO (the CO saturation value at 25 ° in pure water is o.93. lO -3 M). The second order velocity constant thus becomes 72 ± 9 M-1 sec-~. The light reaction is almost insensitive to changes of temperature within the range used in these experiments. But the light intensity has, of course, an influence on Biochim. Biophys. Acta, 54 (1961) 132-144
COMPLEXES OF BACTERIAL CATALASE
139
the rate of the dissociation reaction and on the equilibrium. Fig. 6 shows the effect of a 4-fold variation in light intensity (two-fold varidtion in light source distance) on the rate of dissociation. The equilibrium, expressed as the ratio of the concentrations of light dissociated BMC-Az II to dark associated BMC-Az II-CO was found to be proportional to the light intensity and extrapolation to zero intensity gave an equilibrium value of zero. 0,5
0.4
-o
t
0.3
0.2 o 0.1
0 0 I ( I 0 3 Cr'n- 2 )
Fig. 6. The equilibrium of reaction 3 of the t e x t as a function of light intensity. Ordinate, equili[BMC-Az I I ] A d b r i u m , expressed a s t h e ratio I ° ° - I B M C - A z IIJ = i o o - A d where the concentration of thedissociated form is expressed in per cent of the total catalase concentration, i.e. [BMC-Az II1 + EBMCAz II-CO]. Abscissa, relative light i n t e n s i t y I, expressed as the reciprocal s q u a r e of the distance in lO 3 cm -2.
Circumstantial evidence of the presence of a true equilibrium in Eqn. 3 is provided by an experiment where the dissociation of BMC-Az II-CO by light is periodically interrupted by short dark periods, as shown in Fig. 7. The individual traces of the light reaction can be combined to a composite trace that is identical with that of an uninterrupted light reaction. The light dissociation of the CO compound was repeated in a series of experiments at pH 6. 9 (20 m M phosphate) and gave essentially the same kinetic and equilibrium data as at pH 5.4.
4A
--~--
Expt Ill
Composite B
CurVe -
~
T
O.OSO
Fig. 7. A, an original recording obtained b y interm i t t e n t illumination of BMC-Az I I - C O . To a 0.22 # M solution of BMC in 2o m M p h o s p h a t e buffer, p H 5.4, MeOOH was added to a final concentration of o.67 raM. This w a s followed b y an addition of N a N 3 to a final c o n c e n t r a t i o n of 6.7 m M and b y i rain b u b b l i n g w i t h p u r e CO gas. The difference of a b s o r b a n c y at 428 m/~ a n d 4o6 m/t was m e a s u r e d in the double b e a m spectrop h o t o m e t e r as a function of time. Ordinate, change in a b s o r b a n c y (AA); abscissa, time; i u n i t = i min. The time scale r u n s from left to right. The illuminating light b e a m was t u r n e d on and off as indicated on the record. B, the recording of Fig. 7 A is redrawn, b u t only those p a r t s of the reaction where the light was on are included. The resulting composite curve closely resembles a typical, u n i n t e r r u p t e d light reaction trace such as t h a t of Fig. 5 A.
Biochim. Biophys. Acta, 54 (1961) 132-144
140
A. C. MAEHLY
I t remained to be seen if the light dissociation of BMC-Az I I - C O really leads to BMC-Az II, or if another ~omplex is formed in this reaction. To this end, a light dissociation spectrum was taken b y performing the dissociation and association reactions at a series of wavelengths through the region of the Soret spectrum. As reference wavelength 412 m F was chosen, close to the peak of the expected difference spectrum between the two complexes involved. Since, at the equilibrium in the light, the CO complex is only partially dissociated, partial deflections were recorded. The values thus obtained were compared with the difference spectrum of BMC-Az I I - C O minus BMC-Az I I (recorded on the Yang-Legallais spectrophotometer) b y multiplying them by an arbitrary constant factor. The data in Fig. 8 show that the light dissociation does indeed lead to the formation of BMC-Az II. If intermediates are formed, their lifetime is below the time resolution of the method used here ( ~ I sec). Fig. 8. The difference s p e c t r u m of BMCAz I I - C O m i n u s BMC-Az I I , o b t a i n e d in t w o different ways. I. The u n b r o k e n trace is an original recording (Expt. A 127) o b t a i n e d in the Y a n g - L e g a l l a i s a p p a r a t u s . Conditions: BMC c o n c e n t r a t i o n o . 4 4 / z M in 2o m M p h o s p h a t e buffer, p H 5.4. BMC-Az I I was formed b y the addition of 0.67 m M MeOOH and 6.7 m M azide. Two c u v e t s were filled w i t h the resulting solution. P u r e CO w a s b u b b l e d t h r o u g h one of the c u r e t s for I rain, the c u r e t closed w i t h a greased cover, a n d I i P , r i I ~ P , I , the difference s p e c t r u m recorded in the d a r k 32O 340 360 380 400 420 440 460 480 500 a n d at 25 ° . 2. The open circles represent individual readings in light dissociation exp e r i m e n t s of the t y p e represented in Fig. 5 A. Conditions of the e x p e r i m e n t (A 116) : BMC concent r a t i o n o.16/~M in 20 m M p h o s p h a t e buffer, p H 5.4. The addition of 0.9 m M MeOOH and 6.5 m M azide was followed b y i n t e r m i t t e n t b u b b l i n g of p u r e CO for I-rain periods to form BMC-Az I I - C O . The distance f r o m the illuminating light source was 16 cm. One w a v e l e n g t h of t h e double, b e a m i n s t r u m e n t of CHANCE was set at 412 m/~, for the o t h e r w a v e l e n g t h various values b e t w e e n 360 a n d 460 m # were chosen. At each selected pair of w a v e l e n g t h s the dark a n d / o r light equilibrium values of the a b s o r b a n c y difference were recorded. The whole set of d a t a t h u s o b t a i n e d was plotted in the figure, using an empirical factor for bringing the d a t a to the same scale as t h a t of the recorded difference s p e c t r u m .
Since the experimental conditions for the formation of the CO compound from BMC-Az I I had been clearly established, efforts were now made to demonstrate a CO binding of complex III, both at p H 5.4 and p H 6. 9. In some experiments, complex I I I was formed in the usual way (several additions of small concentrations of H202, Eqn. 2a) and CO bubbled through the solution; in others, the solution of complex I I was saturated with CO and H20 ~ added afterwards. In neither case was there any spectral change observed, either in the dark or during illumination. Working with an anaerobic cuvet 17 in an atmosphere of pure CO, and adding H~O~ from a sidearm gave the same results. In order to see if H~O 2 itself or one of its reaction products (02!) acted as an inhibitor, two kinds of tests were carried out: (a) the formation of BMC-Az I I - C O was allowed to go to completion (dark equilibrium), then successive additions of H~O~ were m a d e - - m o r e than would be needed to form complex I I I from complex II. No spectral change was observed. (b) Conversely, complex I I I was formed in a CO atmosphere. Formation of a CO complex could Biochim. Biophys. Acta, 54 (1961) 132-144
4~
?
~o
~D
el/H
971x, o 4 . 9.5
405 405, 405,
Io2" * * 4o5, x o 2 * * * 406, z o 5 * * *
BMC BMC BMC BMC
--
--
e2/H
5Ol, 9.o*** 505, x z . 4
9.9 ~*~
9.2***
--
--
--
541 , 8 . 6 " * * 538, z o . 2 * * *
545,
5 o6, x o . 7 * * *
545
(536)
536
. 541 ,
--
--
e3/H
536, XT*
54 °
23
. 5o5, t o . 5 ~*~
5°6
503, x 7 . 6 " *
502
505
.~
BANDS
OF
CATALASES
.
edH
63o, 629,
631,
623,
7.z*** 7.2
8.4***
. 7-3***
631 - 626, ( ~ 6)
627, x 2 . 3 " *
624, x o . 6
627
622 -622, x o . 8 623, .rs~ 623, 9
?q
-12.5 14. 4 14.6
13.°
-----
-~2.9 9.5 --__ -13.o
ex e~t
(~) in m/~ ; e / H , e x t i n c t i o n c o e f f i c i e n t p e r h e m i n , in c m -1 × m M - L
ABSORPTION
III
2. 4 H e r o e s . ** 4 H e m e s . * ** C a l c u l a t e d f r o m AGNER'S v a l u e s a n d a s s u m i n g t h a t h e h a d d i v i d e d t h e s e b y 4-
4o5,
95***
405,
405, x 3 8 " 405, x 3 8
405, I 4 2 "
409, x 4 5 4o0, x 4 2 "
2x
HBC HBC HBC HBC HBC HBC
HLC HLC HLC HLC HLC HLC HLC HLC
Type of catalase
Wavelength
TABLE
6.8 -5.4-8.2 - - 7 .2
6.8
7.1 7.I 6.8 --
? ? 7.x 7.1 ---6.0
pH
33 34 This paper 35
29 27 3° 31 32 This paper
2~ 22 23 24 25 26 27 28
Reference
i-4
oq
,.4 t'rl
o~ ©
,.~
©
('3
142
A. C. MAEHLY
not be observed. After addition of 2.5 m M azide, BMC-Az I I - C O was fully formed (dark-equilibrium). These experiments show that only BMC-Az I I but not complex I I I is able to bind carbon monoxide. DISCUSSION
Since both the Soret spectra and the spectra in the visible region were to be compared, a survey of the literature was made in order to find spectral differences in the three most intensively studied types of catalase: horse blood catalase, horse liver catalase, and bacterial micrococcus catalase. In spite of the thousands of publications on these enzymes, very few quantitative spectrophotometric data were found. This is partly due to the lack of adequate spectrophotometers prior to the second World War, partly to the fact that most research was done either in the visible or in the Soret region, but only rarely in both. HERBERT AND PINSENTe calculated their spectral data in the visible range from dry weight determinations of their crystalline bacterial catalase. They found ee31 = 32.4 cm -1 m M -1 (from E % ----- 1.47 ), but gave no values for the Soret region. - - i~cm CHANCE AND HERBERT18 found the extinction coefficient at 405 m ~ to be 405 i 5 cm-1 mM-1, this time based on the measurement of the hemin content of BMC b y forming the pyridin-hemochromogen. The ratio of these two values is ea0Jee31 = 12.5. A direct comparison was made by Dr. A. BRILL b y diluting a stock solution of BMC appropriately with phosphate buffers of p H 5.9-8.2. A ratio eao5/e630 = 14.6 ± 0.2 was obtained. The pyridin hemochromogen method, which was considered more reliable than the dry weight determination, gave a value of e405 = 405 cm -1 m M -1., the same figure as CHANCE AND HERBERT18. I t was taken as the primary standard, and eeso becomes thus 405 :[: 5/14.6 = 28.7 ± 0.3 cm -1 m M -1. The ratio e4oJee30 is higher for BMC than for HBC or HLC. Table I I I gives a review of the published spectral data for the three catalases. It is surprising to see how incomplete these spectral data are in spite of the extent of the literature on catalases. Table I lists the position and intensity of the Soret bands both for the absolute spectra and difference spectra of BMC, BMC-MeOOH II, BMC-H~O, III, BMC-Az I I and BMC-Az II-CO. Some values for HBC and HLC are included. The table proved useful in the choice of appropriate wave lengths for the rate and equilibrium studies with the double beam spectrophotometer. The spectral data for the visible region are compiled in Table I I in the same manner as in Table I. Here a considerable discrepancy is found between the absolute spectra of BMC-Az I I (and its CO complex) and HLC-Az I I (and its CO complex). The spectrum published by KEILIN AND HARTREEle for HLC-Az I I - C O in particular has a very different shape from that observed here for BMC-Az II-CO. This is made clearer by a glance at Fig. 9 which gives the difference spectra of these compounds minus free catalase. Plotted in this way, comparison between HLC and BMC is facilitated. The light dissociation data presented here certainly confirm KEILIN AND HARTREE'S observations, and extend them to include kinetic measurements. However, * I n t h e p u r e s t p r e p a r a t i o n s of BMC, a v a l u e of ~o5 = 420 c m-1 m M - 1 , close t o t h e h i g e s t v a l u e p u b l i s h e d b y DEUTSCH 19 :[or HBC, a n o t h e r 4-heroin c a t a l a s e , w a s o b t a i n e d b y Dr. A.BRILL 2°.
Biochim. Biophys. Acta, 54 (1961) 132-144
143
C O M P L E X E S OF B A C T E R I A L CATALASE
experimental difficulties have so far prevented a calculation of the actual rate and equilibrium constants. These difficulties stem from the competition of 0 , and CO for Cat-Az II. Working under the usual conditions of anaerobiosis, i.e., in an inert gas, does not remove all oxygen from Cat-Az II. Addition of sodium dithionite does not affect the CO compound but decomposes Cat-Az II rapidly. The only experiments where the CO concentration was known were those at complete C0-saturation, achieved by extensive gassing of the solution. Further experimentation will be needed to secure the C0-titration data necessary for computing the true constants for the dark and light reactions. The fact that no CO binding could be observed with catalase complex III under the conditions of the experiments reported here has the obvious limitations of all negative evidence. Until such a CO binding can be demonstrated, there is no reason to postulate Complex III as a ferrous compound. // 50
/ ,,
',,
/
40 // -"" E
30'
,
•
20
/
\~
-[E 10
\
/. -
,,~, C
/ ,'-',',~,
/
tl Ill.# " / / /..
o
o
'
%"
.':-
/,
.
~T~.'::~'"
- zo 1 .
,
,
460
500
540
,
,
580
620
,
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F i g . 9- D i f f e r e n c e s p e c t r a of s o m e c a t a l a s e derivatives in the visible region. The differences between the s p e c t r a of B M C - A z I [ a n d B M C - A z I I - C O , r e s p e c t i v e l y and the s p e c t r u m of free B M C a r e plotted. F o r c o m p a r i s o n , t h e s p e c t r a of H L C - A z I I a n d H L C - A z I I - C O r e s p e c t i v e l y versus the s p e c t r u m of H L C a r e p l o t t e d f r o m t h e p a p e r of KEILIN AND HARTREE 16. C, - - - - , B M C - A z I I vs. BMC. C a t a l a s e concentration in b o t h c u v e t s , 0 . 6 / z M . T e m p e r a t u r e + 2 °. 20 m M p h o s p h a t e b u f f e r of p H 5.4. A d d i t i o n s : 2 m M M e O O H + IO m M N a N 3 t o free BMC. D, - . . . . , B M C A z I I - C O vs. BMC. T h e s o l u t i o n w a s o b t a i n e d b y bubbling CO for I min through solution C. I, X - - X , H L C - A z IT vs. H L C , r e p l o t t e d f r o m Fig. I i n KEILIN AND HARTREE'S p a p e r 16. C a t a l a s e concentration not given, p H 6.8. N 2 a t m o s p h e r e . 2, C) ..... © , H L C A z I I - C O vs. H L C , plotted a s for c u r v e I.
ACKNOWLEDGEMENTS The author wants to thank Dr. B. CHANCE for his interest and encouragement and Dr. A. BRILL for detai]ed discussions. Thanks are due to Mr. L. PAKMAN and Mr. M. BARR for their cooperation in preparing the enzyme. The work was supported in part by a grant from the U.S. Public Health Service, Division of Grants and Fellowships. REFERENCES 1 y . OGURA, H. HASEGAWA, K. NAGANO AND K. OGASAWARA, Symposia on E n z y m e Chem., Japan, 9 (1954) 91. 2 y . OGURA, Arch. Biochem. Biophys., 57 (1955) 288. 3 y . OGURA, p e r s o n a l communication. 4 D. KEILIN AND E . F. HARTREE, Proc. Roy. Soc. (London) B, 121 (1936) 173. Biochim. Biophys. Acta, 54 (1961) 1 3 2 - 1 4 4
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5 D. KEILIN AND E. F. HARTREE, Biochem. J., 49 (1951) 88. 6 D. HERBERT AND J. PINSENT, Biochem J., 43 (1948) 203. 7 R. F. BEERS, Science, 122 (1955) lO16. 8 A. BRILL AND A. C. MAEHLY, to be published. 9 D. HERBERT AND J. PINSENT, Biochem. J., 43 (1948) 193. 10 C. C. YANG AND V. LEGALLAIS, Rev. Sci. Instr., 25 (1955) 8Ol. 11 C. C. YANG, Rev. Sci. Instr., 25 (1955) 807. 12 B. CHANCE, Rev. Sci. Instr., 22 (1951) 634. 13 B. CHANCE, L. SMITH AND L. CASTOR, Biochim. Biophys. Acta, 12 (1953) 289. 14 K. AGNER AND H. THEORELL, Arch. Biochem., io (1946) 321. 15 B. CHANCE, J. Biol. Chem., 194 (1952) 471. 16 D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 148. 17 A. C. MAEHLY, L. SMITH AND J. D. GRAHAM, Jr., Science, 122 (1955) 767. 18 B. CHANCE AND D. HERBERT, Biochem. J., 46 (195o) 402. 16 H. F. DEUTSCH, Aeta Chem. Scan&, 6 (1952) 1516. 2o A. BRILL, personal c o m m u n i c a t i o n . 21 K. G. STERN, J. Gen. Physiol., 20 (1937) 631. 22 K. G. STERN, J. Biol. Chem., 121 (1937) 561. 23 K. AGNER, Biochem. J., 32 (1938) 17o2. 24 K. AGNER, Arkiv. Kemi Mineral. Geol., 16A (1942) No. 6. 25 A. L. DOUNCE AND O. D. FRAMPTON, Science, 89 (1939) 300. 26 j . B. SUMNER, Advances in Enzymology, Vol. i, Interscience Publishers, 1941, p. 163. 27 R. K. BONNICHSEN, Arch. Biochem., 12 (1947) 88. 26 D. KEILIN AND E. V. HARTREE, Biochem. J., 49 (1951) 88. 26 K. AGNER, Arhiv. Kemi Mineral. Geol., I7B (1943) No. 9. 30 D. HERBERT AND J. PINSENT, Biochem. J., 43 (1948) 203. 31 H. F. DEUTSCH, Acta Chem. Scan&, .5 (1951) lO74. 32 H. F. DEUTSCH, Acta Chem. Scand., 6 (1952) 1516. 33 D. HERBERT AND J. PINSENT, Biochem. J., 43 (1948) 193. 34 B. CHANCE AND D. HERBERT, Biochem. J., 46 (195 o) 402. 35 A. BRILL, personal c o m m u n i c a t i o n . 36 y . OGURA AND A. EHRENBERG, results obtained at + 9 ° in a B e c k m a n D U s p e c t r o p h o t o m e t e r a t the Nobel Medical I n s t i t u t e , Stockholm. 37 R. K. BONNICHSEN, Arch. Biochem., 12 (1947) 83. 36 B. CHANCE, Arch. Biochem. Biophys., 41 (1952) 404 . 39 y . OGURA, personal c o m m u n i c a t i o n . 40 H. THEORELL AND A. EHRENBERG, Arch. Biochem. Biophys., 41 (1952) 442. 41 H. THEORELL AND A. EHRENBERG, personal communication. 42 y . OGURA, personal c o m m u n i c a t i o n . 43 D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 148. The e values per h e m i n were multiplied b y 4. 44. H. THEORELL AND K. AGNER, Arkiv Kemi, Mineral. Geol., I6A (1942) No. 7.
Bioehim. Biophys. Acta, 54 (1961) 132-144