Biochimica et Biophysica Acta, 427 (1976) 652-662

(© Elsevier Scientific Publishing Company, Amsterdam ..... Printed in The Netherhmd~. BBA 37298 M A G N E T I C C I R C U L A R D I C H R O I S M STUDIES ON H O R S E R A D I S H PEROXIDASE

TSUNENORI NOZAWA, NAGAO KOBAYASHI and MASAHIRO HATANO Chemical Research Institute of Non-aqueous Sohttions, Tohoku UniversiO, ~Semkti, 9,~'0 ,'Japm~.,

(Received September 8th, 1975)

SUMMARY Magnetic circular dichroism (MCD) spectra were observed lk)r native (Fe(111)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano- derivatives, and also for reduced (Fe(ll)) horseradish peroxidase and its carbonmonoxy- and cyano- derivatives. M C D spectra were obtained lk~r the cyano derivative of Fe(lll) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different M C D spectrum from that of myoglobin hydroxide. Thus it shows an M C D spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical M C D spectra with those fl~r the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heine iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.

INTRODUCTION It has become evident that the magnetic circular dichroism (MCD) is very sensitive to the spin and oxidation state of the heine group from extensive studies of myoglobin, hemoglobin, and their derivatives [1-10]. The M C D spectra of cytochrome c [2, 3, 11-13], b2 [13, 14], b5 [15, 19] as well as heine itself [16-18] have added additional information to the evidence obtained from studies of myoglobin and hemoglobin. A heme enzyme (monooxidase) cytochrome P-450 has been successfully investigated with the aid of M C D [19-22]. However there exists insufficient experimental evidence to deduce the relation between observed MCD and structural concomitants of changes in the electronic state of the heine. Recently resonance Raman spectra of horseradish peroxidase have been reported and a sensitivity claimed to structural differences in the heine iron [23]. This report stimulated us to measure the M C D of horseradish peroxidase. In this paper similarities and differences are discussed between the MCD spectra of horseradish

653 peroxidase and myoglobin derivatives in order to examine the potentiality of the MCD. MATERIALS

AND

METHODS

Horseradish peroxidase (Sigma type II) was purchased and used without further purification. The purity value defined as the ratio of absorbances at 403 and 275 nm, was 1.48. Solutions were prepared in 0.I M phosphate buffer (pH 7.01) at 0 °C. The concentrations were determined from the known molar extinction coefficient for appropriate derivatives [23]. The fluoro- and cyano- derivatives were prepared by adding solid N a F or K C N respectively, until the absorption spectrum changes showed saturation [23]. Horseradish ferroperoxidase was prepared by adding solid sodium dithionite to a solution of the native protein. Its cyano- and carbonmonoxyderivatives were prepared by adding K C N and bubbling CO, respectively, in the presence of excess sodium dithionite. M C D was measured with a JASCO J-20C recording spectropolarimeter equipped with an electromagnet which produces a magnetic field of up to 11.4 kG. The magnetic field was determined with freshly prepared ferricyanide using the established value [0]M~422) = 1.0 [1 ]. The instrument is calibrated using the natural CD of D-10-camphorsulfonic acid as a standard with [0]290 = 7260 [1 ]. The M C D magnitude

474

405 299

" ~

524

615

365

658

-1 550

I

-3

V

I

424 402

-4 -5

A

®

;'69

%

495

1 ×

645 3OO

4OO

/~ (nm)

500

60O

7O0

Fig. 1. MCD and absorption spectra of native Fe(IlI) horseradish peroxidase. Concentration 1.924. 10 -4 M in 0.1 M potassium phosphate buffer (pH 7.01); path = 1 mm below 450 nm, 5 mm above field = 11.4 k G ; t e m p e r a t u r e - - 0 ° C . V e r t i c a l line = c h a n g e o f e scale as i n d i c a t e d b y encircled m a g n i f i c a t i o n factor. 450 nm;

654 was expressed by the molar ellipticity per gauss ([0]M(deg'cm-'-dmol ' - ( ; ~)) on the basis of the molar c o n c e n t r a t i o n of the heine unit. The m e a s u r e m e n t s were carried out at 0 °C. Quartz cells with 5 1 mm path lengths were placed in a quartz Dewar ol ice water. A n a u t o m a t i c slit width affording a spectral bandwidth of less than 2 nm was employed and found sufficient to resolve all bands. Scan rate of 5 nm..min x~ay used in c o n j u n c t i o n with a time constant of 4 sec. RESULTS AND DISCUSSION M C D spectra are shown for native ( F e ( l l l ) ) horseradish peroxidase, its alkaline form, and fluoro- and cyano- derivatives, and also for reduced (Fe(ll)) horseradish peroxidase, a n d its c a r b o n m o n o x y and cyanide derivatives in Figs. 1 7. Nearly identical M C D spectra were observed for the cyano- derivative o1 native horseradish peroxidase, and the c a r b o n m o n o x y derivative of reduced horseradish peroxidase with those for the respective myoglobin derivatives (see Table 1).

V .... T......... .....

5! ~0~

..........

;

/ 29~ ~

463

~

273

- 5~

-10

.................

~

-15 ~

r

('~ b'b

,

o 539

900

z.O0

/~ ( n m ,

BO'J

t 1 ×

cX),]

-,7~), :

Fig. 2. MCD and absorption spectra of an alkaline form of Fe(lll) horseradish peroxidase. Concentration 2.28.10 -4 M in water (pH 11.90); path .... 1 mm below 450 nm, 5 mm above 450 nm; field 11.4 kG; temperature 0 'C. Vertical line = change of e scale as indicated by encircled magnification factor

655 The rest of the derivatives show somewhat different M C D spectra from those for myoglobin (Mb). Above all the alkaline form of the native horseradish peroxidase exhibits a completely different M C D spectrum from that for Mb hydroxide [1, 7]. The alkaline horseradish peroxidase shows a clear A-term-type M C D in the Soret region. Its magnitude is twice as large as that for Mb hydroxide and nearly identical with that for Mb azide (Table I). Similar results were observed in the visible region. Thus, the apparent A term associated with the absorption peak at 577 nm looks like that for Mb azide rather than that for Mb hydroxide. These M C D results indicate either that the alkaline form of native horseradish peroxidase should have a stronger field ligand than O H - , or that the ligand field of the (possibly coordinated) O H - was strengthened for some reason. These results are also consistent with the fact that the magnetic moment of the alkaline form of horseradish peroxidase ( ~ ¢ f f = 2.66fl) [25] is closer to that of Mb azide (/~eff ---- 3.30fl) rather than Mb hydroxide (/tell = 5.04/3) [25]. Together with the fact that an alkaline low-spin form is a full 2 p K a units higher (11.0 compared to 9.0) for horseradish peroxidase [24] than for methemoglobin (Hb) [27], 396 297

599

499

s~'~

369 -2

40~

623

-3 -~

273

403 427

Fig. 3. MCD and absorption spectra of a fluoro derivative of Fe(III) horseradish peroxidase. Concentration approx. 1.11.10 .4 M, below 450 nm, and 4.29.10 .4 M above 450 nm, in 0.1 M potassium phosphate buffer (pH 7.01) with 1.5 M fluoride; path = 1 mm below 450 nm, 5 mm above 450 nm; field = 11.4 kG; temperature 0 °C.

656 it seems that the alkaline form of horseradish peroxidase is not a usual heme hydroxide complex. This is very significant when the differences in native horseradish peroxidase from Mb or Hb are considered. The native horseradish peroxidase exhibits fairly similar MCD spectra to thai for Mb, though the relative magnitudes of the MCD trough around 650 nm was reversed from those tbr the one at 550 nm. A significant difference exists especially m the Soret region. Not only did the Soret MCD magnitude decrease to one half, but also the shape of the shorter wavelength peak changed considerably. It appears that this nmy be due to tile result of an overlap with a positive A term around 395 nm which

i ,] k

o L~---294 . . . . . . . .

487

~Zo I ~

.

.

.

.

.

.

.

.

.

.

.

.

279 -10 ~

-2oL+

I/ I

!iH -30'

~426

x

4,

'o 1

300

,,C,:)

),

(nrn)

S

,,

i~:3 '

~/%

Fig. 4. M C D and absorption spectra of a cyano derivative of Fe(III) horseradish peroxidase. C o n centration approximately 1.81 • 10 -4 M in 0.1 M potassium phosphate buffer (pH 7.01) with 1 mM cyanide; path 1 m m below 450 nm, 5 m m above 450 nm ; field 11.4 kG ; temperature 0 'C.

657

associates with either extra absorption bands or vibrational bands of the Soret transition. Recent results of resonance Raman spectra of native horseradish peroxidase revealed that it is anomalous in that the Raman marker frequencies do not fall into the high-spin Fe(III) grouping [23]. This anomaly has been explained with the assumption that a high-spin native (Fe(III) horseradish peroxidase heme is closer to being planar than the other high-spin Fe(III) heme proteins so far examined. This assumption has been applied to the explanation for differences between the native horseradish peroxidase and Mb or Hb which shows extensive doming of the porphyrin ring upon out-of-plane displacement of the iron atom [27, 28]. As described above, some differences were observed in MCD spectra between native horseradish peroxidase and met-Mb. Yet, the native horseradish peroxidase still exhibits a MCD spectrum characteristic for the ferric high spin grouping rather than for the ferric low spin grouping. Subtle differences in the Soret MCD spectra may be attributable to the domed nature 439

2(

lC

551

~.2

293

/.11 435

15

uO 5

....

, .... 300

, . . . . . . . . .

,

~0 h

j

(rim)

' '~0

. . . . . . . . .

600

, , ' '

.

.

.

.

Fig. 5. M C D and absorption spectra of reduced Fe(II) horseradish peroxidase. Concentration 2.47. 10 -4 M and 0.1 M potassium phosphate buffer (pH 7.01); path = 2 m m below 450 nm, 5 m m above 450 nm; field = 11.4 k G ; temperature = 0 °C. Vertical line = change of [0]ra scale as indicated by encircled magnification factor.

658 of the porphyrin. Therefore it is concluded that the M C D spectra on the whole are more sensitive to the spin state of the heme iron atom rather than the porphyrin structures. This indicates that the electronic interaction between porphyrin and iron does not substantially depend on the position of the iron relative to the heme phme. Since the R a m a n resonance spectrmn of Fe(lll) horseradish peroxidase fluoride was found to fall into a typical high-spin ferric grouping, it seems to have the heine with usual d o m e d conformation characteristic of Hb fluoride [231]. I::e(lll)horseradish peroxidase fluoride exhibits an M C D spectrum almost identical ~ith that of Mb fluoride, although some differences can be seen in the Soret region. Thus, the Sorel M C D magnitude is greater for Fe(lll) horseradish peroxidase fluoride than for metMb fluoride. Moreover, the shape is significantly different. The magnitude and shape of Fe(lll) horseradish peroxidase fluoride spectra look more like that for lnet-Mb water [1 ]. Therefore it is concluded from the M C D for native horseradish peroxidase and its fluoride derivatives that the Soret M C D is more sensitive to the spin state and structures of the heine than the visible one. The M C D spectra are ahnost identical both in shape and magnitude for the reduced horseradish peroxidase and its c a r b o m n o n o x y derivative. Still there are some minor differences. The visible M C D spectra of reduced horseradish peroxidase have 15 L

567

294

- 5~-

277

-15F

V Z'2.

5

300

400

~ ',nm)

500

GO0

~;i3

Fig. 6. MCD and absorption spectra ofa carbonmonoxy- derivative of Fe(ll) horseradish peroxidase. Concentration approximately 6.92.10 -s M, in 0.1 M potassium phosphate buffer (pH 7.01 ): path 2ram; field ll.4kG: temperature 0 C.

659 an MCD peak and trough around 580 and 560 nm very distinct in comparison with those of deoxy-Mb. The ferrous cyanide derivative is obtained only for horseradish peroxidase because its myoglobin or hemoglobin derivative is very unstable [28]. The characteristic features of ferrous low spin hemochrome are observed for the cyanide derivatives of reduced horseradish peroxidase. Thus, it shows distinct Faraday A terms for the Soret, Q0-~, and Q0-0 absorption band. The MCD magnitude of the Q0-0 band is much larger than that for the Soret band, which resembles the oxy-Mb more closely than carbonmonoxy-Mb. The small trough around 438 nm has not been observed for other ferrous low spin heine proteins so far investigated. The MCD spectra below 350 nm were not discussed in this paper because those for Mb derivatives are not still established, and moreover the protein purity of horseradish peroxidase was not high enough to guarantee the spectra in the protein region. CONCLUSION

The MCD spectra of horseradish peroxidase derivatives were found to be essentially similar to those for myoglobin derivatives except for the alkaline form. This implies that MCD is sensitive to the spin and oxidation states of heme rather

10

410

E

312

360

536 422

c'~n

-10

561

5 x eO

® 300

400

500

600

700

Fig. 7. M C D a n d a b s o r p t i o n spectra o f a c y a n o derivative o f Fe(II) h o r s e r a d i s h peroxidase. Concentration 9.65. l0 -5 M a n d 2.03- 10 -4 M below a n d above 450 n m , in 0.1 M p o t a s s i u m p h o s p h a t e buffer (pH 7.01) with approximately 1 m M cyanide; p a t h - 5 m m below 4 5 0 n m , 1 m m above 450 n m ; field -- 11.4 k G ; temperature -- 0 °C. Vertical line = c h a n g e o f e scale as indicated by encircled magnification factor.

660 TABLE 1 ABSORPTION AND MAGNETIC CIRCULAR ROXIDASE AND MYOGLOBIN COMLEXES Derivatives

Valence state o f iron

Ligand

Fe(lll)

H20

Fe(lll)

Alkaline [orm

OF THE HORSERADISH

Horseradish peroxidase

Myoglobin ~

Absorption

MCD

Absorption

2 ......

~ • 10 - )

}.

[0]~

2 ......

~-10

(rim}

( c m . M ) -~

(nm}

degcm 2 /drool • G

(nm)

(cm.M)

409

157

402

91

495

10

645

Fe(lll}

DICHROISM

416

2.8

88

539

8.6

575

6.9

Fluoride 403

125

558

5

620

7

391 405 413 424 474

0.8 1.(} (} 2.9 1.2

'

9.47

635

3.55

0.4 I. I 0.3 0.2 0.4

405 41 t 420 463

15 (} 21 1.5

414

97.2

526 560 580 642

1.6 1.8 2.8 0.3

542

9.5

582

9.1

369 396 404 408 427 449 471 499 523 544 571 599 624

t.2 3.1 {} 1.8 3.7 1.5 1.2 O.25 0.4 0.16 0.44 0.88 1.9

MC1)

505 524 550 580 615 658

406

133

490

8.3

609

7.8

PE

'

}.

[#1~,

(nm}

,..leg.era: ,.Imol. (~

4i)4 413 429 4~4 4S3 52O 533 550 590 (~14 (~4(~ v~(,7 412 421 42s ~45 4~7 49{1 ~3{} 550