291

Biochimica et Biophysica Acta, 577 (1979) 291--306 © Elsevier/North-Holland Biomedical Press

BBA 38141

MAGNETIC C I R C U L A R DICHROISM STUDIES OF BOVINE L I V E R CATALASE

WILLIAM R. BROWETT and MARTIN J. STILLMAN * Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7 (Canada)

(Received August 1st, 1978) Key words: Catalase; Magnetic circular dichroism; Heme protein

Summary Absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra o f beef liver catalase at pH 5.0 and 6.9, and its complexes with NaF, KCNO, NaCNS, NaN3 and NaCN, have been measured b e t w e e n 250 nm and 700 nm at r o o m temperature. The pH 6.9 native catalase MCD shows the presence of several additional transitions not resolved in the absorption spectrum. While these bands can be seen in the spectra of all the derivatives, with the exception of the cyanide, their relative intensities changes considerably between complexes. Of special interest in the MCD o f ferric hemes is the signal intensity at a b o u t 400 nm and 620 nm. The data indicate that the MCD intensity at 620 nm increases as the high spin iron porphyrin fraction increases, reaching a maxim u m with the fluoride complex. The 430 nm band intensity increases as the proportion of low spin iron increases, reaching a maximum with the cyanide complex. The MCD spectra also indicate clearly the existence of spin mixtures in the complexes with CNO-, CNS- and N3-, where both the 430 nm and 620 nm bands have appreciable intensity. It is significant that despite almost identical absorption spectra the CNS- complex has a higher fraction of low spin iron than either the CNO- or the N3- species. The differences b e t w e e n the pH 5 and 6.9 MCD spectra of the native catalase suggest that the environment o f the heme centre is sensitive to protonation.

* To whom correspondence

should be addressed.

292 Introduction Catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) is a heine protein which contains an iron protoporphyrin IX prosthetic group. Physiologically catalase is believed to function as a hydrogen peroxide scavenger, reducing and oxidizing hydrogen peroxide to water and oxygen. With only a few exceptions, only strict anaerobes have been found to lack this enzyme [1]. Though there are reported variations all catalases appear to be oligomers containing four tetrahedrally arranged 60 000 dalton subunits. Each subunit contains a single polypeptide chain that associates with a single heme [2]. A thorough review of catalase and the nature of its active site, and catalase mediated redox reactions has recently appeared [2]. Of primary importance amongst the reactions of catalase are those involving peroxides, the 'catalatic' reaction [2,3,4]. Native ferric catalase + H202 -~ C o m p o u n d I C o m p o u n d I + H202 -* ferric catalase and the 'peroxidatic' reaction: Native ferric catalase + H202 -* C o m p o u n d I C o m p o u n d I + AH -~ C o m p o u n d II + A" C o m p o u n d II + AH -~ ferric catalase + A" Neither the molecular geometry nor the electronic configuration of the iron porphyrin moiety in either C o m p o u n d I or II are known. However, by analogy with the related peroxidases and porphyrin radical-cation complexes [5] it has been suggested that Compound I is a Fe(IV) porphyrin cation radical complex, while C o m p o u n d II is an Fe(IV) porphyrin. A more detailed description of the electronic structure of the heme in catalase is necessary for the elucidation of the active site and the mechanism of the enzymatic function. Comparison of the chemical and physical properties of catalase with other heme proteins, such as the peroxidases, is essential if the characterization of its active site is to be achieved. We may then be able to determine the local environment criteria that result in this protoporphyrin IX group exhibiting such a unique chemistry. To this end, data from spectroscopic techniques that are sensitive to the heine group that is the active site in catalase may be directly compared to data measured for the well known heme proteins such as hemoglobin, myoglobin, horseradish peroxidase and some of the cytochromes. A variety o f spectroscopic techniques have been used to study catalases, such as, absorption [6,7], Raman [8], NMR [9--11], EPR [12--19], CD [20] and recently MCD [ 21]. MCD provides a sensitive non-destructive probe o f the electronic structure of the heme. MCD spectra of several other heme proteins, such as cytochromes [22--37], myoglobin [28--32], hemoglobin [33], and peroxidase [34,35], illustrate well the techniques' sensitivity to the electronic configuration of the iron porphyrin complex. The MCD technique is especially sensitive to transitions in which there is a

293 large change in orbital angular momentum. The metalloporphyrin that comprises the heme group in catalase has an absorption spectrum that is dominated by transitions involving the 181r electron system of the ring [36,37]. Under Dah symmetry, the states linked by these transitions have been calculated to have significantly different orbital angular momentum values, approximately 1 unit in the Soret band and 4 units in the a band [38]. MCD studies of a range of metalloporphyrins [39] and phthalocyanines [40] indicate that these numbers are reasonable. Thus the MCD spectrum provides an additional parameter with which the spectrum may be characterized. There are three specific spectral line shapes observed in a normal MCD spectrum. (i) A derivative shape with a cross-over point lying under the band centre of the absorption band identifies a degenerate excited state, this is the Faraday A term. (ii) A Gaussian-like band of either sign with the same band centre as the absorption band, with an intensity that increases with decreasing temperature, identifies an orbitally degenerate ground state, this is the Faraday C term. (iii) A Gaussian-like band of either sign that has a temperature independent intensity, arises from magnetically induced mixing of all available excited states, this is the Faraday B term. In view of the complexity of the room temperature spectra of ferric heme proteins, the assignment of any transition as either an A term or B/C term is very .difficult. In addition, the close proximity of states of similar orbital origin results in significant mixing, which means that the terminal states of these transitions lose their pure character, and therefore the angular momentum associated with the degenerate 7r system is now averaged over many more transitions. Reviews of MCD spectra and theory have appeared [41--44]. The spectra shown in this study were obtained only at room temperature thus the B and C terms are indistinguishable. Magnetic susceptibility, absorption and EPR data [3] all indicate that catalases exist as high spin d s iron complexes at physiological pH. Catalase shows little change in absorption spectra from pH 4.7 to 9.0, though horse erythrocyte catalase exhibits a high to low spin transition at pH 11 as the temperature is dropped from 300 K to 77 K [6]. The heme iron of native catalase at pH 7.0 appears to be six coordinate with a proposed fifth coordination position, Ls, possibly being filled by a histidine amino acid [2,7] and a sixth coordination position being filled by a water molecule [9,10]. The ligand exchange reactions at this position, L~, have a stoichiometry expressed by the reaction PrFe(III)H20 + AH ~ PrFeAH + H 2 0 , where Pr represents the protein backbone, and Fe(III) the iron porphyrin complex. This has been shown to be a second order reaction which is dependent upon the acid/base equilibrium of the ligand: AH~-A- +H ÷ , between pH 4.0 and 9.0 [45]. Though A- is thought to be the coordinating species there is no net up-take or release of a proton. A model for the active site has been proposed [2].

I

/H

I

Ls--Fe--O~

+ AH ~ Ls--Fe--AH + H20 YH H

294 It is assumed that Ls does n o t change with ligand exchange. Unlike other heme proteins catalase has a greater tendency toward the formation of high spin complexes which may indicate that the heme in catalase may exist in a protein environment of low dielectric constant [46] or the protein environment moderates the ability of the anionic ligands to complex with the heme. MCD spectra have been obtained in this study of a variety of derivatives in order to study the effects of pH and ligand exchange on the heme chromophore of beef liver catalase. Our analysis attempts to correlate the observed spectral changes with changes in the electronic structure of the iron and porphyrin at the active site.

Experimental Bovine liver catalase was obtained from Boehringer Mannheim as a crystalline suspension in 0.01% alkylbenzyldimethyl a m m o n i u m chloride with an apparent rate constant o f 2.0 • 107 s-1 • M -1 in phosphate buffer, pH 7.0, I = 0.05. All chemicals were Fisher Certified ACS grade, with the exception o f KCNO which was Allied Chemical Reagent ACS grade. Catalase solutions were prepared by dialysis of the crystalline suspension twice against water triply distilled from alkaline permanganate solution and twice against phosphate buffer, pH 7.0, I = 0.05. The solutions were heated to 37°C to dissolve the enzyme, cooled to room temperature and centrifuged or filtered when necessary to remove any suspended material. The pH 5.0 and pH 8.8 catalase solutions were prepared by dialysis o f aliquots o f the pH 7 solution against 0.02 M lactate and phosphate buffers (I = 0.05) respectively. The purity index of the catalase solutions was measured spectrophotometrically as A4os/A276, this value varied between 0.88 and 0.89 for all solutions for which spectra are shown. The ligand exchange complexes were obtained by adding sufficient solid sodium or potassium salt until the absorption spectrum indicated a single species had been formed. These spectra were compared with those formed previously by titra= tion. Enzyme concentration determinations were based upon the use of an extinction coefficient of l 0 s dm 3 • mo1-1 • cm -1 per heme at 404.5 nm [47]. The absorption spectra were obtained using a CARY 14 spectrophotometer and a combination of either 0.10 cm and 0.5 cm, or 0.2 cm and 1 cm cells in order to measure both the Soret and visible regions with the same solution. The CD and MCD spectra were obtained on a JASCO-UV-5 spectrometer with a Sproul conversion to a SS-20 CD-ORD spectrometer. This i n s t r u m e n t was wavelength calibrated to the CARY 12t using holmium (III) (Coming) and neod y m i u m (III) (JASCO) filters. The CD spectra are reported in terms of molar eUipticities, [0], with units deg" dm 2" dmol -i. The spectrometer was calibrated with a freshly prepared solution of (+)-10-camphor sulfonic acid with a concentration o f 1 mg/ml using 0 2 9 1 n m = 0.308 °. MCD spectra were obtained using the 5.5 T field of an Oxford Instruments Magnet. The MCD spectra are reported as molar ellipticities per unit tesla, [0 ] M, with units deg - dm 2 • dmol -I • T -I. The field strength and sign were checked by measuring the 510 nm MCD spectrum of aqueous CoSO4, [0]M at 510 nm for our instrument was calculated to be --59.3 d e g . dm 2- dmo1-1- T -1. Catalase solutions with a m a x i m u m

295

absorbance of 1.6 at 405 nm in a 1 cm cell were used together with a scan speed of less than 15 nm per min. With the exception of the MCD peak at 294 nm associated with tryptophan [48] all spectra are believed to be fully resolved. The peak at 294 nm is relatively intense and extremely narrow and it may be under resolved by about 10% under the sampling conditions which were used to obtain the spectra from 300--700 nm. The spectral uncertainty in the MCD data was less than +5% after subtraction of the CD data. Absorption spectra were obtained for each solution used in the MCD experiment to check the spectral purity and enzyme stability. All spectra shown were automatically digitized using a digital sampling unit attached to either the Jasco or the CARY 14 wavelength drive and slidewire (Scheiring, M.J. and Stillman, M.J., unpublished). Digitized copies of each spectrum were generated on paper tape as the spectrum was recorded. The MCD spectra were obtained by digital subtraction of the zero-field spectrum from the field-on spectrum for each solution. The data presented here are retraced computer plots. The ordinate scales of the MCD and CD spectra differ only in the magnitude of the magnetic field, thus division of the CD scale by 5.5 T and addition to the MCD spectrum will regenerate the original spectrum recorded with the 5.5 T field. Results

The absorption, MCD and CD spectra of bovine liver catalase obtained at pH 5.0 and 6.9 together with complexes formed by ligand exchange with NaF, NaCNS, KCNO, NaN3 and NaCN for the spectral region 250--700 nm are shown in Figs. 1--7. The spectral data is summarized in Table I. The native catalase spectrum is characterized in the absorption spectrum by distinct charge tranfer transitions at 622 nm and 500 nm, a Soret band at 404.5 nm and a protein band absorption maximum at 276 nm which arises from the aromatic amino acids present. The purity of the enzyme is often measured as the ratio of absorbance A4os/A276, our samples had a value of 0.88 which can be compared to reported values for beef liver catalase of 0.82 [14] and 0.84 [21], while the purity number for horse erythrocyte catalase is between 0.9 and 1.0 [12] and for bacterial catalase the range is 1.0 to 1.2 [7]. Beef liver catalases are often contaminated by bile pigment [1,2,16]. The bile pigment is believed to arise from the degradation of a hematin group in the molecule during extraction. The activity of the enzyme has been found to be directly proportional to the hematin concentration and inversely proportional to the iron concentration associated with biliverdin [1]. Though the bile pigment contamination may affect the absorption spectrum, the affect on the MCD spectrum, which is much more sensitive to the higher D4h symmetry of the porphyrin ring, should be limited to a weak, broad B term. The MCD spectrum of bilirubin, a product of the degradation of bile pigment [49], showed negligible intensity throughout the spectrum. The absorption spectra of bovine liver catalase at pH 5 and pH 6.9 are almost identical, Figs. 1 and 2, and indeed, there is almost no change in this spectrum when the pH is changed from 4.7 to 9.0 [2,6]. However, there is a significant difference between the MCD spectra of the pH 5.0 lactate buffer solution, Fig. 2, and the pH 6.9 phosphate buffer solution, Fig. 1. The pH 6.9 MCD spectrum

TABLE

I

CATALASE

MCD, CD AND AM C D

p H 6.9

ABSORPTION

[0] M X 10 -3

388 403 429 485 531 540 555 614 628 643

0 --19 --15 12 7 0 --16 9 0 --14

405 432 483 532 556 609 626 640

--18 --7" 14 7 --10 10 0 --19

394 411 421 440 459 502 564 589 606 615 625

--5 4 --7 16 10 6 --5 19 8 0 --28

395 404 432 482 530 608 623 639

0 --11 --13 13 8 11 0 --20

382 397 414 428 482 531 612 622 638

0 13 7 --39 10 7 7 0 --22

369 389 409 421 431 484 530 612 623 637

--16 0 18 0 --20 13 7 11 0 --25

p H 5.0

F-

CNO-

CNS-

N~

SPECTRAL ACD 291 348 388

290 345 390

295 335 393

291 342 390

DATA [0] X 10 --4 8.0 0 --7.9

6.6 O --8.2

4.6 0 --9.0

7.2 0 --8.2

AABS

e X 10 -3

404.5 494 544 622

100.0

404.5 494 544 622

100.0

8.77

8.80

406

99.7

596

12.7

408.5

101.7

494 535 621 292 340 392

7.7 0 --7.0

408.5

9.03

101.8

494 535 621 291 345 390

9.20

6.3 0 ---6.9 411

99.8

494 535 620

9.76

297 TABLE I(continued)

k MCD

[0] M X 1 0 - 3

kCD

CN374 418 426 434 490 548 572 580 602

293 350 394

0 85 0 --111 12 9 --I 0 --17

[ 0 ] X 10 -4

k ABS

e X 10 -3

9.1 0 ---8.4 423

76.5

554 578

11.4

is characterized by a broad, derivative-shaped band envelope centred o n the absorption band maximum at 6 2 2 nm. While this MCD band might arise from a single transition, its broadness, and the shoulder at approx. 6 2 0 nm for both the pH 6.9 and pH 5 data, and the presence o f multiple bands in the fluoride spectrum, Fig. 3, all suggest that the envelope comprise a series o f overlapping bands. To higher energy, the m u c h sharper signal at 540 nm has the typical A-term characteristics associated with the a band o f the ~ -~ lr. spectrum, while

h

ABSORPTION

10C $ 5(;

1.5

'

46o

'

5;o

'

6&

' MCD

a.

=05 -05

5"5

CD l

i

6bo

'

-5'5

3bo '

,~o

'

~o

'

WAVEL.ENGTH/nm Fig. 1 . T h e a b s o r p t i o n , M C D a n d C D s p e c t r a o f n a t i v e c a t a l a s e i n p h o s p h a t e b u f f e r , p H 6 . 9 , I = 0 . 0 5 . T h e a b s o r p t i o n s p e c t r u m b e t w e e n 2 6 0 - - 7 0 0 n m w a s o b t a i n e d u s i n g a s o l u t i o n h a v i n g A 4 0 s of 0 . 9 i n a 0 . 1 c m cell. T h e c o r r e s p o n d i n g visible r e g i o n s p e c t r u m 5 0 0 - - 7 0 0 n m w a s o b t a i n e d u s i n g a 0 . 5 c m c e l l . T h e e x t i n c tion coefficient has units of mM -I • cm -I . The MCD and DC spectra were obtained using a solution h a v i n g A 4 0 s o f 1 . 6 i n a 1 c m cell.

298

ABSORPTION

% c 50'

400

1-~

50O

600

&

"~oe -0"~

5

'

5

~

300

400

500 600 WAVELENGTH /nm F i g . 2. T h e a b s o r p t i o n , M C D a n d CD s p e c t r a o f c a t a l a s e a t p H 5 . 0 in a 0 . 0 2 M l a c t a t e b u f f e r . T h e a b s o r p t i o n s p e c t r u m b e t w e e n 2 5 0 - - 7 0 0 n m w a s o b t a i n e d u s i n g a s o l u t i o n h a v i n g A405 = 0 . 8 5 in a 0 . 2 c m cell. T h e c o r r e s p o n d i n g visible r e g i o n s p e c t r u m , 5 0 0 - - 7 0 0 n m , w a s o b t a i n e d u s i n g a 1 c m cell. T h e M C D a n d C D s p e c t r a w e r e o b t a i n e d u s i n g a s o l u t i o n h a v i n g a n A 4 0 5 = 1 . 4 i n a 1 c m cell.

\

ABRPTN

5(2

1-~

500

A

• T

= 0"~ -0.~

-5

" 300

5

~

400 500 WAVELENGTH/nm

600

Fig. 3. T h e a b s o r p t i o n , M C D a n d C D s p e c t r a o f c a t a l a s e p l u s 0 . 5 M N a F i n a 0 . 0 2 M l a c t a t e b u f f e r , p H 5 . 0 , o b t a i n e d b y a d d i t i o n o f N a F t o t h e s o l u t i o n s w h o s e s p e c t r a are s h o w n i n Fig. 2. T h e s p e c t r a l eondi~ir~n~

299

in the Soret region there are t w o prominent negative transitions at 429 nm and 403 nm. An intense, narrow line at 294 nm dominates the 250 nm--300 nm region in every MCD spectrum. Assigned as a t r y p t o p h a n transition [48] its intensity acts as a useful guide to the relative intensities o f the heme group bands. Upon changing the pH to 5.0 there is significant increase in the [0]M intensity at 640 nm, the region of the spectrum normally associated with charge transfer transitions [36] and a corresponding decrease in [0]M at 555 nm, the and /3 transition region. While the charge transfer region around 500 nm remains stable, there is a strong decrease in intensity of the negative band at 431 nm in the Soret region. The corresponding spectra at pH 8.8 in a phophate buffer were found to be identical to those obtained at pH 6.9 except for a slight increase in intensity of the low energy shoulder in the charge transfer region of the spectrum at 660 nm. The spectral results of the ligand exchange complexes are shown in Figs. 3--7. We have assumed in our arguments that the presence of the ligand at the high concentrations (approx. 104 molar excess of added ligand) needed to ensure complexation [7,45], does n o t cause a change in the coordinating species at the fifth position of the heme. This is confirmed by our observation of isosbestic points on titration o f NaCN with native catalase at pH 7, which indicate that only t w o species are present, the aquated iron and the low-spin iron b o u n d to the cyanide.

ABSORPTION 100

OCN

-

50.

1"~

" 0.5

]

400

500

6(20

-

-0.5 -1.5

5

T

5

300

A

.

4O0 500 WAVELENGTH/nrn

CD

600

F i g . 4. T h e a b s o r p t i o n M C D a n d C D spectra o f e a t a l a s e p l u s 0 . 0 5 M K O C N i n a p h o s p h a t e buffer, p H 6 . 9 , I = 0 . 0 5 o b t a i n e d b y a d d i t i o n o f K O C N to a n a l i q u o t o f t h e s o l u t i o n s u s e d to o b t a i n the spectra s h o w n in F i g . 1.

300

IO0/~ 5C

ABSORPTION

'

4~X) '

5~3

'

6&

'

: 05

-0.5 CD

- 5-5 38o

'

46o

'

56o

'

WAVELENGTH/nm

66o

Fig. 5. T h e a b s o r p t i o n MCD a n d CD s p e c t r a of c a t a l a s e plus 0 . 0 5 M N a S C N in a p h o s p h a t e b u f f e r , p H 6.9, I = 0 . 0 5 o b t a i n e d b y a d d i t i o n o f N a S C N t o a n a l i q u o t o f t h e s o l u t i o n s u s e d t o o b t a i n t h e s p e c t r a in Fig. 1.

ABSORPTION

-~

%~ 10050--

500

1.5

600

--0.I -1 .!

-5

.

300

5

~

4O0 500 600 WAVELENGTH/rim

Fig. 6. T h e a b s o r p t i o n , M C F , a n d CD s p e c t r a of catalase p l u s 0 . 0 5 M N a N 3 in a p h o s p h a t e b u f f e r , p H 6.9, I ffi 0 . 0 5 o b t a i n e d b y a d d i t i o n o f N a N 3 t o a n a l i q u o t o f t h e s o l u t i o n s u s e d t o o b t a i n t h e s p e c t r a s h o w n in Fig. 1.

301

10(:/,~

ABSORPTION

,

'

40

CN-

'

5~9

'

6~)0

' MCD

-1.! CD

I

I

I

|

5OO WAVELENGTH/nrn

6OO

Fig. 7. T h e a b s o r P t i o n , M C D a n d C D s p e c t r a o f c a t a l a s e p l u s 0 . 0 1 M N a C N in phosphate buffer, p H 6 . 9 , I = 0 . 0 5 obtained by addition o f N a C N t o a n a l i q u o t o f t h e s o l u t i o n s u s e d t o o b t a i n t h e s p e c t r a i n F i g . 1.

The catalase fluoride complex which was prepared for these spectra in a pH 5.0 lactate buffer may also be prepared by addition of a large excess of NaF at pH 7 in a phosphate buffer with no apparent difference in the resultant MCD spectra. The absorption spectrum of the fluoride complex is characterized by a shift of the charge transfer band at 622 to 596 nm and an apparent loss in detail associated with other transitions observed in the visible region of the absorption spectrum. In the Soret region the absorption spectrum shows only a slight reduction in intensity upon complexation. The corresponding MCD spectrum is characterized by a large reduction in [8]M intensity in the Soret region together with a wavelength shift of all the bands in the spectrum relative to the pH 6.9 catalase spectrum. The electronic changes in the charge transfer band at 596 nm due to the fluoride binding to the iron result in a large increase in MCD intensity. The band envelope is now observed to be clearly split into two transitions. Though the CNO- and CNS- derivatives have remarkably similar absorption spectra there is a very significant difference in the MCD of the two species in the Soret region. In both complexes the Soret absorption band shifts to 408 nm from the 404.5 nm of native enzyme, while in the visible region there is a slight increase in absorption and a shift of the charge transfer band to 621 nm. The MCD of the CNS- complex shows a dramatic increase in intensity under the Soret band at 428 nm relative to the CNO- spectrum. Indeed, this is the

302 most intense Sorer band MCD observed for any of the species that exhibit a high spin spectrum. The N3- spectrum has an absorption spectrum characteristic of a high spin complex, although the shift of the Soret band to 411 nm suggests an increase in low spin component. The MCD in the visible region is very similar to the spectrum obtained for the CNS- complex, though the charge transfer band at 637 nm is n o w 20% more intense. However, the Soret band shows a large change in shape and intensity relative to the pH 6.9 catalase species, with a strong positive peak at 409 nm and a strong negative trough at 431 nm. Catalase forms a low-spin ferric complex with CN- characterized b y both EPR [ 18] and magnetic susceptibility results [3,36]. The absorption spectrum shows a reduction in Soret band intensity and a shift to 423 nm compared with the native enzyme. There is also a reduction of intensity in the charge transfer regions at 500 and 620 nm together with a simultaneous increase in intensity associated with the ~ and fi transition region. The ~ band is still a poorly resolved shoulder on the side of broad fi band. The MCD spectrum is characteristic of other low-spin d s h e m e ~ y a n i d e spectra. Those obtained for myoglobin [28] and peroxidase [34] represent typical spectra. The MCD in each case is dominated in the Soret region b y an intense, narrow, positive A term; for catalase the negative trough is at 434 nm, the cross-over at 426 nm and peak at 418 nm. In the visible region there are strong MCD transitions associated with the a and bands. The 620 nm charge transfer bands have negligible intensity, while the 500 nm charge transfer band becomes indistinct and merges with the ~ band envelope. The CD spectra which have been obtained for these species show a much less dramatic variation either with pH or ligation and little correspondence to the changes which occur in the absorption or MCD spectra. The CD spectra for all the derivatives are characterized by a positive rotation at 290 nm, a crossover at 345 nm, and a negative rotation at 390 and 450 nm. The latter two peaks become more distinct on complexation with N3- or CN-. It is well known that the CD spectral intensities under the heme group transitions depend u p o n the conformation of the protein backbone rather than a chirality of the heme group itself [50,51]. As these CD spectra are almost invariant to ligand type, it is probable that the catalase protein conformation does not alter significantly between each species. In addition, while these very broad CD bands m a y coincide with some of the weak and overlapping bands in the absorption spectra between 300 and 450 nm, it appears that they do not arise from the same transitions that generate most of the MCD intensity. The MCD bands are much narrower and their band centres vary considerably from species to species. Thus the MCD spectrum is sensitive to the local heine environment, while, independently, the CD spectrum is sensitive to the protein conformation. Discussion

The MCD data indicate that catalase in the ferric oxidation state is predominantly high-spin in nature as previously observed using b o t h absorption and EPR spectroscopy. Though a first coordination sphere of the iron has been pro-

303

posed which is similar to the active site in myoglobin, hemoglobin and horseradish peroxidase, there is a much larger high-spin component in the catalase complexes. This difference does not necessarily exclude the possibility of the 5th and 6th coordination positions being filled by a histidine amino acid residue and H20 respectively, but could represent a difference in interaction induced by the protein environment close to the heme. While catalase shows no significant spectral change between pH 4.7 and 9.0 by absorption and EPR, there is a considerable change in the MCD spectrum upon reduction of pH from 6.9 to 5.0. Assuming that this change is not associated with a ligation change, it must represent a subtle change in the heme environment, such as the protonation of an amino acid or the conformational change of the heme cleft. By exchanging the axial ligand of the iron it is possible to form complexes that change from high to low-spin ferric iron. Typical ligands used throughout heme protein chemistry to illustrate the effect of spin on the porphyrin spectrum are F- for high spin, N3- for intermediate spin, and CN- for low spin iron. The major spin changes in the iron are readily observed with EPR and magnetic susceptibility techniques. In the protons used here as examples, ferric myoglobin fluoride is characterized by a bulk paramagnetic susceptibility moment of 5.76 BM * [36] and the appearance of change transfer bands in the 610 and 804 nm regions [36]. Similarly, ferric horseradish peroxidase fluoride exhibits a series of spectral properties characteristic of a high spin ferric porphyrin, a magnetic moment of 5.92 BM and charger transfer bands are observed in the near-infrared [4]. Catalase fluoride also has spectral properties that indicate its high spin nature, EPR values for horse erythrocyte catalase show greatest intensity at g = 6.6 and 5.4 and only a minor band at g = 2.3 [13], a magnetic moment of 5.78 BM [12] and charge transfer bands at 598 and 820 nm [13]. Although the EPR and absorption spectra of the pH 6.9, pH 5 and fluoride species are similar, the MCD of these three indicates that there is a shift of angular momentum from the Soret region to the 620 nm charge transfer bands, as we go from pH 6.9 to pH 5 and then to the fluoride complex, Figs. 1--3. While this effect is relatively slight for the pH 6.9 to 5 spectra, the fluoride spectrum shows a considerable redistribution of angular momentum into the 600 nm region. The 8oret band magnitude is much reduced compared with the pH 6.9 spectrum. The fluoride MCD, therefore, illustrates another parameter that can be used in the determination of the spin state of the iron and the overlap of the iron d orbitals with the ~ system of the ring. Presumably, the excited states of the a and ~ transitions between 500 nm and 540 nm have now been considerably mixed into the charge transfer states at 600 nm, the angular momentum of 4 to 5 Bohr magnetons associated with the a band [38] being mixed as well, giving rise to the increased MCD signal magnitude at 600 nm. The azide complex has been well studied [12], and it appears that there is a high to low spin transition as the temperature is reduced. This effect is also observed for horseradish peroxidase, but here the degree of low spin character is higher at room temperature if the azide concentration is high [52]. The N3* BM: a b b r e v i a t i o n for B o h r m a g n e t o n , for d e f i n i t i o n see C o t t o n , F . A . and Wilkinson, G. (1972) Advanced Inorganic C h e m i s t r y , 3rd edn., p. 537, Wiley Interseience.

304 catalase complex absorption spectrum, Fig. 6, is almost the same as the native enzyme in the visible region, b u t the Sorer band shifts 6 nm to 411 nm. The red shift of the Soret band has been used in the past as a rough indicator of low-spin character: here, for example, the Soret band for pH 5 catalase is at 404 nm, fluoride at 405 nm, azide at 411 nm and cyanide at 423 nm. The MCD spectrum clearly contains a low-spin c o m p o n e n t in the Soret region. The increase in the magnitude of the envelope at 420 nm may be considered to arise from an A term centred under the low energy side of the Soret band representing the low spin species band centre. Compare this spectrum with that of catalase CN-, Fig. 7, and the low spin ferric horseradish peroxidase cyanide and hydroxide of Nozawa et al. [34]. A similar effect has also been observed for myoglobin derivatives [28,30]. The cyanate derivative has a much less pronounced Sorer band MCD than either CNS- or N3-. While the CNS- and N3- spectra are in some respects very similar, the intense negative lobe at 430 nm in the CNS- spectrum suggests a greater low-spin component. It is possible therefore, to use the sensitivity of the MCD spectrum to the spin state to derive an order for increasing low-spin c o m p o n e n t as: CNO- < N3- ~ CNS- < CN-. The spectral complexity in the Sorer region and the relatively low-spin character of the CNS- and N3- complexes in comparison to analogous myoglobin complexes, prevents an ordering of the low-spin components. However, it is clear that the catalase derivatives are significantly more high-spin than the corresponding myoglobin complexes. The extreme low-spin complex of catalase which is formed with NaCN is in fact more comparable to the myoglobin N3- complex in spectral characteristics and intensity than the corresponding myoglobin CN- complex. The differences may be attributed to either a smaller low-spin c o m p o n e n t in the catalase CN- complex or possibly a greater rhombicity o f the heine [17]. Thus, unlike other heine proteins, the iron in beef liver catalase does not appear to bond as strongly to the complexing ligand. This effect may be caused b y either the necessity of the formation of a complex between the conjugate acid form of the ligand and both the heme iron and an adjacent amino acid residue, in the manner described by Schonbaum and Chance [2], or the strong interaction of the protein with the heme b y the proximal and distal amino acids that creates an environment that results in a predominantly high spin iron porphyrin with most axial ligands and a significant proportion of high spin iron even with cyanide as a ligand. The recent low temperature MCD results from ferrous oxymyoglobin that shows that below 100 K there is a 10 to 20% high spin ferrous c o m p o n e n t [30] lends support to the argument that a protein is able to moderate the effective ligand field of the ligand. The tendency of a heme toward a high spin iron in the porphyrin has been associated with the square pyramidal conformation of the heme [53] which is stabilized b y weakly coordinating solvents. In a very recent paper Blum e t a l . [54] have observed the conversion between two rhombic forms of beef liver catalase heme using EPR spectroscopy. The fact that the most rhombically distorted form is stable below pH 5, while a less distorted configuration exists above pH 7 correlates well with o u t MCD spectra of the 620 nm change transfer and 400 nm ~r -* 7r. bands that indicate that at pH 5 the heme has a higher spin than at pH 7. It was further suggested that this pH change which has a strong influ-

305 ence on the local symmetry of the heme is associated with a histidine side chain. The strong interaction of the protein with the ligated heme that prevents the iron from feeling the full ligand-field effect is in keeping with the model of the amino acid side chain interaction proposed by Schonbaum and Chance [2]. In addition, the protein environment of the heme only allows the interaction of protonated ligands or neutral peroxides. An electrostatic gate hypothesis has been proposed by Davies et al. [55] where negatively charged groups within the active site could control access to the heme. Unionized ligands would then be able to diffuse to the heme while anions would b e excluded. Ionization within the active site would allow the anionic form of the ligand to bind to the heme [56]. Acknowledgement Financial support by the National Research Council of Canada and by the Research Corporation through a Cottrell Grant is gratefully acknowledged. References 1 Deisseroth, A. and Dounce, A.L. (1970) Physiology Reviews 50, 319--375 2 Schonbaum, G.R. and Chance, B. (1976) in The Enzymes (Boyer, P.D., ed.), 3rd edn., pp. 363--408, Academic Press, New York 3 NichoUs, P. and Schonbaum, G.R. (1964) in The Enzymes (Boyer, P.D., Lardy, H. and Myrback, K., eds.), 2nd edn., Vol. 8, pp. 147--225, Academic Press, New York 4 Dunford, H.B. and s t i n m a n , J.S. (1976) Coord. Chem. Rev. 19,187--251 5 Dolphin, D. and Felton, R.H. (1974) Aec. Chem. Res. 7, 26--32 6 Yoshida, K., lizuka, T. and Ogura, Y. (1970) J. Biochem. 68, 849--857 7 Brill, A.S. and Sandberg, H.E. (1968) Biophys. J. 8,669---690 8 Felton, R.H., Romans, A.Y., Yu, Nai-Teng and Schonbaum, G.R. (1976) Biochim. Biophys. Acta 434, 82--89 9 Lanir, A. and Schejter, A. (1975) FEBS Lett. 55, 254--256 10 Lanir, A. and Schejter, A. (1976) Biochemistry 15, 2590--2596 11 Hershberg, R.D. and Chance, B. (1975) Biochemistry 14, 3885--3891 12 Torii, K., lizuka, T. and Ogttra, Y. (1970) J. Biochem. 68, 837--841 13 Torii, K. and Ogu~ra, Y. (1968) J. Biochem. 64, 171--179 14 Torii, K. and Ogttra, Y. (1969) J. Biochem. 65, 825---827 15 Williams-Smith, D.L. and Morrison, P.J. (1975) Bioehim. Biophys. Acta 405, 253--261 16 WilHams-Smith, D.L. and Patcl, K. (1975) Biochim. Biophys. Aeta 405, 243--252 17 Blumberg, W.E. and Peisach, J. in Oxidases and Related Redox Systems (King, T.E., Mason, H.S. and Morrison, M., eds.), Vol. 1, pp. 299--310, University Park Press, Baltimore 18 Rein, H., Ristau, O., Hackenberger, F. and Jung, F. (1968) Biochim. Biophys. Acta 167,538--546 19 Ristau, O., Rein, H. and Hackenberger, F. (1970) FEBS Lett. 9, 71--72 20 Samejima,T.and Kita, M. (1969) Bioehim. Biophys. Acta 175, 24--30 21 Kajiyoshi, M. and Anan, F.K. (1977) J. Biochem. 81, 1319--1325 22 Brlttain, T., Sprlngall, J., Greenwood, C. and Thomson, A.J. (1976) Biochem. J. 159, 811--813 23 Shimizu, T., Nozawa, T., Hatano, M., Imai, Y. and Sato, R. (1975) Biochemistry 14, 4172--4178 24 Dolingex, P.M., Kielczewski, M., TrudeU, J.R., Barth, G., Linder, R.E., Bunneberg, E. and Djerassi, C. (1974) Proc. Natl. Acad. Sei. U.S. 71,399--403 25 Dawson, J.H., TrudeU, J.R., Barth, G., Linder, R.E., Bunnenberg, E., Djerassi, C., Chiang, R. and Hager, P. (1976) J. Am. Chem. Soc. 98, 3709--3710 26 Dawson, J.H., Holm, R.H., Truden, J.R., Barth, G., Linder, R.E., Bunnenberg, E., Djerassi, C. and Tang, S.C. (1976) J. Am. Chem. Soc. 98, 3709--3710 27 Vickery, L., Nozawa, T. and Sauer, K. (1976) J. Am. Chem. Soe. 98, 351--357 28 Vickery, L., Nozawa, T. and Sauer, K. (1976) J. Am. Chem. Soe. 98, 343--350 29 Sharonov, Y.A., Mineyev, A.P., Livshitz, M.A., Sharonov, N.A., Zhurkin, V.B. and Lysov, Y.P., (1978) Biophys. Struct. Mech. 4, 139--158

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