Biochimica et Biophysica Acta, 493 (1977) 340--351
© Elsevier/North-Holland Biomedical Press BBA 37728 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 ACID A N D A L K A L I N E FORMS OF H O R S E R A D I S H PEROX1DASE
NAGAO KOBAYASHI, TSUNENORI NOZAWA and MASAHIRO HATANO* Chemical Research Institute of Non-aqueous Solutions, Tohoku University, Sendai 980 (Japan)
(Received January 18th, 1977)
SUMMARY The heme vicinities of the acid and alkaline forms of native (Fe(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (Ili) protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentacoordinated heme complex. The MCD spectra of native horseradish peroxidase did not show any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.
INTRODUCTION Recently magnetic circular dichroism (MCD) spectroscopy has been extensively applied to various hemoproteins, such as myoglobin, hemoglobin [1-10], cytochrome c [2, 3, 11-13], cytochrome b2 [13, 14], cytochrome b5 [15, 16], cytochrome P-450 [17-19], and cytochrome oxidase [20-23]. The results indicated that the magnetic circular dichroism is a powerful technique to verify the oxidation, spin and ligand states of heme chromophores. MCD spectra of native (Fe(III)) and reduced (Fe(II)) horseradish peroxidase and their derivatives were reported in recent papers [24]. It demonstrated that most derivatives of horseradish peroxidase had MCD spectra similar to those of respective Author to whom correspondence should be addressed. Abbreviations: CD, circular dichroism; MCD, magnetic circular dichroism.
341 myoglobin derivatives [24]. However, the alkaline form of native horseradish peroxidase (at pH 12.01) showed a peculiar MCD spectrum different from that of metmyoglobin. This difference has been pointed out from magnetic measurements and absorption spectroscopy [25, 26]. That is, the alkaline form of native horseradish peroxidase has smaller magnetic susceptibility value (/~eff = 2.66fl) than that of metmyoglobin (~er~ z 5.04fl) and its absorption spectrum resembles that of a low spin type. In spite of a diversity of proposals [27-32], no established result has been offered concerning the kind of ligands existing above and below the heme iron. In this report MCD spectra of native horseradish peroxidase from the acid form (pH < 7) to a peculiar alkaline form (pH > 12) were finely measured and attempts were made to deduce the 5th and the 6th ligands by examining changes in electronic states. EXPERIMENTAL PROCEDURES Horseradish peroxidase (Sigma type II) and myoglobin (Sigma type II) were purchased and used without further purification. The purity defined as the absorptivity ratio at 403 nm and 275 nm, was 1.48 for the horseradish peroxidase. Cytochrome c (Saccharomyces oviformis) was supplied from Sankyo Pharmaceutical Co. Ltd. Absorption spectra were measured at 0 °C in a Hitachi EPS-3T spectrophotometer, using cuvettes of 10mm (700-1800nm), 5 mm (450-700nm) and 1 mm (250-450 nm) optical pathes. MCD spectra were recorded on a JASCO J-20A (2501000 nm) and a JASCO J-200 (1000-1800nm) spectropolarimeter equipped with JASCO electromagnets to produce longitudinal magnetic fields up to 15 000 gauss at samples. Magnetic field was determined with freshly prepared ferricyanide using the established value; [0]M (422nm) 1.0 [1]. The magnet was operated in a normal mode producing the longitudinal magnetic field parallel to the direction of the light. To separate natural circular dichroism (CD) from each MCD spectrum, the spectra were recorded twice; once with magnetic field parallel and then without any magnetic field. MCD magnitude was expressed by the molar ellipticity per gauss ([0]M; degree cm2/dmol gauss) on the basis of the heme concentration. Samples were prepared in a 0.1 M phosphate buffer (pH 7.01), then pH was adjusted to the desired pH values by adding concentrated sodium hydroxide or hydrochloric acid solution. A cell of 5 mm (450-700 nm) or 1 mm (250-450 nm) path and concentration of 2 to 4/zM were used for measurements in the near ultraviolet and visible region; the cell was placed in a quartz Dewar vessel containing ice-water. For measurements in the near infrared region a 10 mm cell was used at concentrations of 0.4 to 0.9 mM in a 0.15 M deuterium oxide phosphate buffer at ambient temperatures. An automatic slit width affording spectral band width of less than 2 nm was found sufficient to resolve all bands. Scan rate of 5 or 10 nm/min was used with a time constant of 4 s. =
RESULTS Fig. 1 shows the MCD and absorption spectra of native horseradish peroxidase. The MCD and absorption spectra remained unchanged in the pH range from 5.20 to 9.00. The MCD spectra in this pH range are characteristic of ferric high spin hemoproteins. The visible MCD spectra show two sinusoidal curves; the larger one consists of a trough at 550-551 nm and a peak at 524 nm with a crossover at
342
........ pH 5.20 2
- - I 3117.01 406
!
299
-474
- - - p H 9.00
^
0 E
550"I -2 -3
424
1.0
4O2
-4
~
-5
x
496
300
&O0
500
35
600
uJ
700
~(nm)
Fig. 1. MCD and absorption spectra of native (Fe(IlI)) horseradish peroxidase in an acid form. Concentration, 1.924.10 -~ M in 0.1 M potassium phosphate buffer (pH 7.01); path length 1 mm below 450 nm, 5 mm above 450 nm; magnetic field 11 400 gauss; temperature 0 °C. Vertical line = change of e scale as indicated by magnification factor. 535 nm, based mainly on the Q absorption band, and the smaller one has a trough at 658 nm and a peak at 615 nm with a crossover at 626 nm associated mainly with charge-transfer bands [1, 28]. A larger apparent A term in the Soret region is composed of a trough at 424 nm and a peak at 406 nm with a crossover at 414 nm. Fig. 2 illustrates the Soret M C D spectra at various p H values corresponding to the M C D changes associated with the transition from acid to alkaline forms. The 424 nm trough developed its intensity with an increase of p H to [0]M of --22.4 at p H 12.01. The peak at 406 nm of the two peaks in the Soret region was predominant in th¢ higher p H region (pH > 11.10) resulting in the M C D peak with [0]M = 16.4 at p H 12.01. The intensity of the Soret trough of the alkaline form ([0]M = --22.4) for the native horseradish peroxidase is far bigger than that observed for metmyoglobin hydroxide ([0]M = --8.3) which is evidence that the alkaline form of the native horseradish peroxidase is composed of more low-spin species than that of metmyoglobin. This is consistent with the result obtained from magnetic susceptibility and absorption spectra [25, 26]. The magnitudes of the Soret M C D spectra at the positive and negative extrema are plotted as a function of p H in Fig. 3. Both the peak at 406 nm and the trough near 424 nm increased throughout the p H range measured, indicating a conversion from high to low spin complex. The midpoint for the transition observed occurred at p H 11.0, which is in good agreement with the results previously reported [25-29, 32]. Assuming that M C D recorded at p H 7 corresponds to the 100% acid form and
343
pH 12.01 ,pH 11.30 ,pH 11.10 ,pH 10.80 ,pH 10.50 .pH 10.25 _DH 10.00
10
0
-I0
-20
MCD at v a r i o u s pHs
400 /~(nrn )
450
Fig. 2. Soret MCD spectra of Fe(III) horseradish peroxidase corresponding to the transition from acid to alkaline forms. Concentration, 2.28.10 -4 M in water; path length 1 m m ; magnetic field 11 400 gauss; temperature 0 °C. ^.alkaline -form acid form
.~,~. ~ , (,n
--
O
~
Sorer 0
[e]m
Peak co
c~
p. . . .
r,D
'o
"0 -r
tog acid form Soret Trough [O]M alkaline form Fig. 3. M C D spectrophotometric titration of horseradish peroxidase. The data are taken from Fig. 2.
344
403
- - Nb(lll')-H20
295
- - - ; ge(X) Protoporphyrin IX Dimethyl Ester CHsCO0-
2
,r,\,
/'l
I I
567
~o 517
-I
64~ -2 -3 -4 ;21 -5
~
503 502
633
300
400
500
600
x
700
A (nm)
Fig. 4. M C D and absorption spectra of metmyoglobin acid form ( p H 7.01) and the metmyoglobin carboxylate complex. Concentration, 2.81.10-4 M for both derivatives; path length 1 mm below 450 nm, 5 mm above 450 nm; magnetic field 11 400 gauss; temperature 0 °C. M C D of acetato-iron (III) p r o t o p o r p h y r i n IX dimethyl ester was replotted from data of ref. 33.
that formed at pH greater than 12 to the 100K alkaline form, we plotted the ratio of the acid to the alkaline form as a function of pH in Fig. 3. The pK value of 11.0 is calculated from either extremum for the transformation of acid to alkaline form. No slope of the data from pH 5 to 9 in Fig. 3 might reflect the absence of any intermediate complexes throughout this region at 0 °C. Fig. 4 shows the MCD and absorption spectra of metmyoglobin carboxylate together with those of the acid metmyoglobin [1] and a carboxylate complex of Fe(III) protoporphyrin IX dimethyl ester [33] from published data. The acid form of horseradish peroxidase gave an MCD spectrum somehow different from that of acid metmyoglobin. The visible MCD spectrum of metmyoglobin carboxylate is strikingly similar in shape and magnitude to that of acid metmyoglobin.
345
Figs. 5 and 6 illustrate the MCD and absorption spectra in the near infrared region for acid and alkaline forms of native horseradish peroxidase, and a cyanide complex of native horseradish peroxidase, ferric cytochrome c at p2H 6.86 and 11.90, I
I
I
,,,
i ,, ..},,,
I
i f HRP -;p2H 6.86
',,,
,,"
,,,
]/]//"
...... ;p2H12.10
'\
"
o x
ol
700
!_
I
8(30
I
900
I
1000
I
II00 1200
.... * .....
I
1300
1400 1500
k(nm)
Fig. 5. M C D and absorption spectra of Fe(III) horseradish peroxidase ( ) and its alkaline form (. . . . . ) in the near infrared region. Concentration 4.64.10 -+ M in 1/15 M deuterium oxide phosphate buffer (p2H 6.86) for native horseradish peroxidase and 3.74' 10 -4 M in deuterium oxide (p2H 12.01) for the alkaline form; path length 10 ram, magnetic field 14 700 gauss; temperature 20 °C. The bars indicate the average noise levels. I
I
I
I
1
I
I
I
I
I
; HRP(]]])-CN.........: Cyt,c( p~H6.86) - . - ; Cyt,c(pRll.90)
/'"t~"~-"~
....
"+
-
I
3
-
~
-
MCD
2o x
\ .L 6 ~4 ~J
2 I
)0
I~"~'1
900
I
II00
I
I
1300 ~.(nm )
I
I
1500
I
I
1700
"7
1900
Fig. 6. M C D and absorption spectra of Fe(IIl) horseradish peroxidase cyanide complex (-), cytochrome c (pZH 6.86) ( . . . . . . ), cytochrome c (p2H 11.90) ( - - - - - ) , metmyoglobin imidazole (. . . . ) in the near infrared region. Concentrations; 3.98.10 -+, 8.60.10 -4, 2.00.10 -3 M in 1/15 M deuterium oxide phosphate buffer (p2H 6.86) for native horseradish peroxidase cyanide complex, cytochrome c (p2H 6.86), metmyoglobin imidazole, respectively, 8.60.10 -+ M for cytochrome c alkaline form in deuterium oxide (p2H 11.90). The cyanide and imidazole complexes were obtained with addition of excess solid potassium cyanide and imidazole, respectively. Path length 10 mm except for metmyoglobin imidazole (2 mm); magnetic field 14 700 gauss except for metmyoglobin imidazole (11 400 gauss); temperature 20 °C, the bars indicate the average noise levels. MCD of metmyoglobin imidazole was replotted from data of ref. 10.
346 [
I
i ~ l i ~ r f l l r
I
5 1 I l l 1
2
-1 -2 --3
-
Cytochrom¢C pH 11.0
---
,,
~
// f
4
---o---HRP Alkatin¢ form pill2,0
/"" i /
/,.
--4
V I
,
~
450
~
J
I
500
t
~
J
I
I
550 3,(nm)
i
L
I
600
~
L
~
f
I
650
Fig. 7. MCD spectra of cytochromec (pH 11.0and 7.0), cytochromeb5and horseradish peroxidasealkaline form (pH 12.0). The data of cytochromes are replotted from data of ref. 15. and metmyoglobin imidazole. The acid form of horseradish peroxidase exhibited troughs at 890 and 1270 nm and peaks at 760 and 1060 nm being somehow similar to that of the acid metmyoglobin [10]. The alkaline form of native horseradish peroxidase showed a peak at 1100 nm with a shoulder on the shorter wavelength side. Fig. 7 demonstrates the visible MCD spectrum for the alkaline form of horseradish peroxidase together with those for some other hemoproteins as low spin models, i.e., cytochrome c at pH 7.0 and 11.0, and cytochrome bs from published data [15]. All these MCD spectra show "S shaped" bands around 540-600 nm, and bell shaped positive bands from 460-530 nm which have been ascribed to the Q and charge-transfer bands, respectively [1]. These charge-transfer bands could not be resolved by electronic absorption spectra, but were clearly detected by MCD spectra. These bands have been assigned to the transition from porphyrin b2u and a'2u er orbitals to iron dn (eg) orbitals [1, 10]. DISCUSSION
The acid form of native horseradish peroxidase exhibited on the whole the MCD spectra peculiar to high spin hemoproteins. Some complex features in the lower wavelength side of the Soret band were discussed in the previous paper [23]. Two different types of visible MCD patterns have been observed among high spin hemoproteins with a histidine as the 5th ligand: one is acid metmyoglobin and metmyoglobin carboxylate, and the other is metmyoglobin fluoride and the high spin component of metmyoglobin hydroxide. This difference has been ascribed to the nature of the 6th ligand of metmyoglobin derivatives [1]. The MCD spectra in Fig. 1 indicate that the acid form of native horseradish peroxidase can be grouped into the high spin hemoproteins of the acid metmyoglobin type. This is consistent with implications from photometric [34, 35] and ESR studies [36] that the proximal ligand is a nitrogen-containing group, probably histidine. However, there exist some fine differences in the intensity ratio of the sinusoidal MCD spectra around 540 nm to
347 that of 640 nm between the acid form of native horseradish peroxidase ( ~ 2) and the acid metmyoglobin ( ~ 1/2). As to this intensity ratio the carboxylate complex of Fe(III) protoporphyrin IX dimethylester in Fig. 4 exhibited a similar ratio ( ~ 2) to that of the acid form of horseradish peroxidase [33]. Since this complex has a pentacoordinated heme, the similarity in the intensity ratio might suggest an electronic structure of the iron for the acid form of native horseradish peroxidase similar to that formed in the pentacoordinated heme. This may be consistent with proton magnetic resonance evidence which suggests the absence of iron-coordinated water in horseradish peroxidase [30]. (However, it should be added that a different interpretation has been proposed for the proton magnetic resonance result [31]). On the other hand, the resonance Raman spectrum of the acid form of native horseradish peroxidase was reported to be different from acid methemoglobin, and this was interpreted as a result of less doming of the porphyrin ring for the former relative to the latter [37]. Therefore the difference in the MCD intensity ratio of the 540 to 640 nm band between the acid form of native horseradish peroxidase and acid metmyoglobin may relate to these structural differences of the porphyrin ring. The visible MCD spectra especially for ferric high spin derivatives are very sensitive to the electronic structures of both porphyrin and iron, because it depends on the complex configuration interactions between the porphyrin Q state and the charge-transfer state associated with iron dn (eg) orbitals. The electronic states of the heine iron depend sensitively on the position of the iron in the porphyrin ring (i.e., doming structure). This position may be determined by the ligand field strength and balance of the apical ligands, and protein structures which control the position and ligand field of the apical ligand atoms. Hence the similarity in the MCD intensity ratio between the acid form of horseradish peroxidase and acetato-iron(III) protoporphyrin IX dimethyl ester may suggest that similar electronic states of the heme iron are formed between the two by the similarity in the iron positions. In the near infrared region the acid form of native horseradish peroxidase exhibited the MCD spectrum which can be grouped into the high spin type of acid metmyoglobin instead of that of metmyoglobin fluoride. However the acid form of native horseradish peroxidase gave an MCD inflection point at 1200 nm in longer wavelength by 100 nm than that of the acid metmyoglobin [10]. The relative magnitudes of the peak to the trough were also reversed between them. These results suggest that, though the acid horseradish peroxidase has a fairly similar ligand state to that of acid metmyoglobin, there exist some fine differences between them. These are consistent with the discussion from the MCD spectra in the Soret and visible regions. Structures of the alkaline form of native horseradish peroxidase have attracted many investigators. It has been established at least that the alkaline form of native horseradish peroxidase belongs to low spin hemoproteins [25, 26, 38]. This is clearly different from the fact that the alkaline form of metmyoglobin has more fraction of high spin component ( ~ 65 ~) than that of low spin component ( ~ 35 ~) at ambient temperatures [28, 39]. Many ideas have been proposed as to the kind of axial ligands of the alkaline form of native horseradish peroxidase [27-32]. The MCD spectral results are consistent with those previously reported in that it clearly displayed the MCD spectra of hemoproteins containing mainly low spin heine chromophores. Furthermore it furnished some evidence to determine the kinds of axial ligands as described below.
348 The MCD magnitude of the Soret band for the ferric hemoproteins has been established to relate closely to the low spin components present [1]. Since the MCD magnitude ([0]~) depends on both the magnetic moment and the electric transition moment of the molecules or ions, the value ([0]M/emM, where emM is the millimolar extinction coefficient), which depends essentially on the magnetic moment, has been proposed to be useful as a spectral index of the MCD spectra [40-43]. Hence the magnitudes of the peak to trough in the Soret MCD bands divided by the millimolar extinction coefficients of the Soret peaks were calculated to be 0.44, 0.34, 0.31, 0.49 and 0.70 for the alkaline form of native horseradish peroxidase, ferric cytochrome bs, metmyoglobin imidazole, and ferric cytochrome c at pH 6.4 and that at pH 11.5, respectively. Ferric cytochrome b5 and metmyoglobin imidazole are the reference hemoproteins with two histidinyl imidazoles as axial ligands. Ferric cytochrome c at pH 6.4 and that at pH 11.5 are the models for the hemoproteins having histidinyl imidazole and methionyl sulfur, and histidinyl imidazole and lysinyl nitrogen, respectively. The calculated values indicate that the axial ligands of the alkaline form of horseradish peroxidase are different from that of ferric cytochrome c at pH 11.0, i.e., the pair of histidinyl imidazole and lysinyl nitrogen. It shows fairly a close value to that of ferric cytochrome c at pH 6.4, but it is not so different from that of ferric cytochrome bs or metmyoglobin imidazole. The visible MCD patterns shown in Fig. 7 also gave a similar conclusion. Fig. 7 shows that the MCD spectra of the alkaline form of native horseradish peroxidase resemble in shape to that of ferric cytochrome c at pH 6.4, but a band shift by ~ 15 nm is noticeable. Though it somehow resembles in shape and magnitude to that of ferric cytochrome bs, a band shift is also noticeable. Furthermore, contrary to the bis-imidazole type complex whose MCD peak at Q0-0 band (around 560 nm) surpasses the charge-transfer bands (470-530 nm) [1] in magnitude, the alkaline form of native horseradish peroxidase maintains the same MCD intensity in these two regions. The alkaline form gives somehow different MCD spectra from that of ferric cytochrome c at pH 11.0 in both shape and band position. So far no definite conclusion can be drawn from the MCD spectra in the Soret and visible regions. However the near infrared MCD spectra gave further implication to determine the axial ligands of the alkaline form of native horseradish peroxidase. As we have discussed in detail for metmyoglobin derivatives in the previous paper [10], the MCD observed in the near infrared region especially for ferric low or high spin derivatives is due to charge-transfer transitions from the porphyrin occupied alu and a2u 7r to iron d~ (e~) orbitals [10, 28]. The alkaline form of native horseradish peroxidase, metmyoglobin imidazole, and ferric cytochrome c at p2H 6.86 and that at pZH 11.90 exhibited similar positive MCD peaks associated with the absorption peaks in the near infrared region. These positive MCD bands have been observed for other ferric low spin derivatives such as metmyoglobin cyanide, the cyanide complex of native horseradish peroxidase (Fig. 6), metmyoglobin imidazole, metmyoglobin azide, and the low spin component of metmyoglobin hydroxide [10]. Although all ferric low spin derivatives displayed similar MCD patterns, there exist substantial differences in the absorption wavelengths. Though the absorption wavelength depends on many factors because these bands are charge-transfer bands mentioned above [10, 28], the overall positions of the absorption bands seem to depend on the kind of axial ligands. It is noticed that the band positions of the near
349 infrared MCD spectra show correlation to the so-called spectro-chemical series. Thus, the MCD peak of the cyanide derivative exists at the longest wavelength (1530 nm), those of imidazole (1520nm), azide (1230nm) and the low spin component of hydroxide complexes (990 nm) following in this order from the longer wavelength. It is very notable that the alkaline form of native horseradish peroxidase exhibited the near infrared MCD peak at 1100 nm was completely different from those of metmyoglobin imidazole (1520 nm), ferric cytochrome c at p2H 6.86 (1540 nm) and ferric cytochrome c at p2H 11.90 (1520 nm). Hence the near infrared MCD spectra indicate that the axial ligands of the alkaline form are different from the pairs of histidine and histidine, histidine and methionine, and histidine and lysine. Among the ferric low spin derivatives studied, the low spin component of metmyoglobin hydroxide showed the near infrared MCD peak at the closest wavelength (990 nm) to that of the alkaline form of native horseradish peroxidase (1100 nm), though it still shows the MCD peak at shorter wavelength by 110 nm. Since, as described above, the near infrared bands shift to a longer wavelength as the ligand field of apical ligands increases, a histidine and a hydroxide ion with some interactions to strengthen the ligand field seem to be a possible pair of apical ligands of the alkaline form. A similar structure has been proposed for the alkaline form of horseradish peroxidase by Yamazaki et al. [27] i.e., the heme vicinity with a hydroxyl group whose basicity at the oxygen is strengthened by some reasons possibly by a hydrogen bonding with the distal histidine. Naturally this is, of course, a possibility. However, this does not exclude other possibilities, such as a pair of a histidine and some other ligand with overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide whose near infrared MCD peaks exist at 990 and 1230 nm, respectively. It may be mentioned here that the alkaline form of horseradish peroxidase exhibited clearly different MCD spectra in the Soret and visible regions, irrespective of the similarity in the near infrared region just mentioned. We need to stress here the fact that the alkaline form resembles the low spin component of metmyoglobin hydroxide. As mentioned earlier, metmyoglobin hydroxide is composed of about 65 ~ high spin and 35 ~o low spin components in thermal equilibrium at ambient temperatures. Although, in the Soret and visible regions, the contribution of the high spin component greatly affects the observed MCD spectra, if we extract the MCD component of the low spin fraction, it will be found that the similarity between the alkaline form and the low spin component of metmyoglobin hydroxide is still held in the Soret and visible regions. In conclusion the acid form of horseradish peroxidase has on the whole a similar electronic structure to that of acid metmyoglobin. However, fine differences between them were ascribed to the differences in the electronic states of the heme iron derived from structure of the heme unit, such as doming of the porphyrin ring induced by strength and balance of the apical ligands associated with protein structures. The alkaline form of native horseradish peroxidase exhibited somehow different MCD spectra from those of typical low spin hemoproteins. The near infrared MCD spectra implicated that the alkaline form seems to have a pair of axial ligands possessing a histidine and another ligand with overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.
350 ACKNOWLEDGEMENTS The authors would like to express their t h a n k s to Professor Isao Y a m a z a k i of H o k k a i d o University for valuable discussions. The authors also wish to t h a n k the Mitsubishi Scientific F o u n d a t i o n for the financial support to construct the near infrared C D spectropolarimeter.
REFERENCES 1 Vickery, L. E., Nozawa, T. and Sauer, K. (1976) J. Am. Chem. Soc. 98, 343-350 2 Schooley, D. A., Bunnenberg, E. and Djerassi, C. (1965) Proc. Natl. Acad. Sci. U.S. 53,579-589 3 Dratz, E. A. (1966) Ph.D. Thesis, University of California, Radiation Laboratory Report UCRL17200 4 Kobayashi, H., Shimizu, M. and Fujita, I. (1970) Bull. Chem. Soc. Japan 43, 2335-2341 5 Djerassi, C., Bunnenberg, E. and Elder, D. L. (1971) Pure Appl. Chem. 25, 57-90 6 Garnier, A., Bolard, J. and Danon, J. (1972) Chem. Phys. Lett. 15, 141-143 7 Bolard, J. and Gamier, A. (1972) Biochim. Biophys. Acta 263, 535-549 8 Yoshida, S., Iizuka, S., Nozawa, T. and Hatano, M. (1975) Biochim. Biophys. Acta 405, 122-135 9 Cheng, J. C., Osborne, G. A., Stephens, P. J. and Eaton, W. A. (1973) Nature 241,193-194 10 Nozawa, T., Yamamoto, T. and Hatano, M. (1976) Biochim. Biophys. Acta 427, 28-37 11 Greenwood, C. and Wilson, M. T. (1971) Eur. J. Biochem. 22, 5-10 12 Sutherland, J. C. and Klein, M. P. (1972) J. Chem. Phys. 57, 76-86 13 Risler, J. L. and Groundinsky, O. (1973) Eur. J. Biochem. 35, 201-205 14 Briat, B., Berger, D. and Leliboux, M. (1972) J. Chem. Phys. 57, 5606-5607 15 Vickery, L. E., Nozawa, T. and Sauer, K. (1976) J. Am. Chem. Soc. 98, 351-357 16 Dolinger, P. M., Kielczewski, M., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E. and Djerassi, C. (1974) Proc. Natl. Acad. Sci. U.S. 71,399-403 17 Dawson, J. H., Dolinger, P. M., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E. and Djerassi, C. (t974) Proc. Natl. Acad. Sci. U.S. 71, 4594-4597 18 Vickery, L., Salmon, A. and Sauer, K. (1975) Biochim. Biophys. Acta 386, 87-98 19 Shimizu, T., Nozawa, T., Hatano, M., Imai, Y. and Sato, R. (1975) Biochemistry 14, 4172-4178 20 Palmer, G., Babcock, G. T. and Vickery, L. E. (1976) Proc. Natl. Acad. Sci. U.S 73, 2206-2210 21 Babcock, G. T., Vickery, L. E. and Palmer, G. (1976) J. Biol. Chem. 251, 7907-7917 22 Thomson, A. J., Brittain, T., Greenwood, C. and Springall, J. (1976) FEBS Lett. 67, 94-98 23 Stillman, M. J., Hollebone, B. R. and Stillman, J. S. (1976) Biochem. Biophys. Res. Commun. 72, 554-559 24 Nozawa, T., Kobayashi, N. and Hatano, M. (1976) Biochim. Biophys. Acta 427, 652-662 25 Keilin, D. and Hartree, E. F. (1951) Biochem. J. 49, 88-104 26 Theorell, H. and Ehrenberg, A. (1951) Acta Chem. Scad. 5, 823-848 27 Yamazaki, I., Hayashi, Y., Makino, R. and Yamada, H. (1975) "Proceedings of the Japan and U.S.A. Seminar, Hawaii", in the press 28 Smith, D. W. and Williams, R. J. P. (1970) Structure and Bonding 7, 1-45 29 Williams, R. J. P., Wright, P. E., Mazza, G. and Ricard, J. R. (1975) Biochim. Biophys. Acta 412, 127-147 30 Lanir, A., and Schejter, A. (1975) Biochem. Biophys. Res. Commun. 62, 199-203 31 Vuk-Pavlovic, S. and Benko, B. (1975) Biochem. Biophys. Res. Commun. 66, 1154-1159 32 Iizuka, T., Ogawa, S., Inubushi, T., Yonezawa, T. and Morishima, I. (1976) FEBS Lett. 64, 156158 33 Dawson, J. H., Holm, R. H., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E., Djerassi, C. and Tang, S. C. (1976) J. Am. Chem. Soc. 98, 3707-3708 34 Brill, A. S. and Sandberg, H. E. (1968) Biophys. J. 8, 669-690 35 Brill, A. S. and Sandberg, H. E. (1968) Biochemistry 7, 4254-4260 36 Yonetani, T., Yamamoto, H., Erman, J. E., Leigh, J. S. and Reed, G. H. (1972) J. Biol. Chem. 247, 2447-2455 37 Gopa, R. and Spiro, T. S. (1974) Biochemistry 13~ 5317-5323
351 38 39 40 41 42 43
Tamura, M. (1971) Biochim. Biophys. Acta 243, 249-258 Iizuka, T., and Kotani, M. (1969) Biochim. Biophys. Acta 181, 275-286 Buckingham, A. D. a/ad Stephens, P. J. (1966) Annu. Rev. Phys. Chem. 17, 399--432 Schatz, P. N. and McCaffery, A. J. (1969) Q. Rev. Chem. Soc. 23, 552-584 Kaito, A., Tajiri, A. and Hatano, M. (1975) J. Am. Chem. Soc. 97, 5059-5062 Kaito, A., Tajiri, A. and Hatano, M. (1976) J. Am. Chem. Soc. 98, 384-388
© Elsevier/North-Holland Biomedical Press BBA 37728 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 ACID A N D A L K A L I N E FORMS OF H O R S E R A D I S H PEROX1DASE
NAGAO KOBAYASHI, TSUNENORI NOZAWA and MASAHIRO HATANO* Chemical Research Institute of Non-aqueous Solutions, Tohoku University, Sendai 980 (Japan)
(Received January 18th, 1977)
SUMMARY The heme vicinities of the acid and alkaline forms of native (Fe(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (Ili) protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentacoordinated heme complex. The MCD spectra of native horseradish peroxidase did not show any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.
INTRODUCTION Recently magnetic circular dichroism (MCD) spectroscopy has been extensively applied to various hemoproteins, such as myoglobin, hemoglobin [1-10], cytochrome c [2, 3, 11-13], cytochrome b2 [13, 14], cytochrome b5 [15, 16], cytochrome P-450 [17-19], and cytochrome oxidase [20-23]. The results indicated that the magnetic circular dichroism is a powerful technique to verify the oxidation, spin and ligand states of heme chromophores. MCD spectra of native (Fe(III)) and reduced (Fe(II)) horseradish peroxidase and their derivatives were reported in recent papers [24]. It demonstrated that most derivatives of horseradish peroxidase had MCD spectra similar to those of respective Author to whom correspondence should be addressed. Abbreviations: CD, circular dichroism; MCD, magnetic circular dichroism.
341 myoglobin derivatives [24]. However, the alkaline form of native horseradish peroxidase (at pH 12.01) showed a peculiar MCD spectrum different from that of metmyoglobin. This difference has been pointed out from magnetic measurements and absorption spectroscopy [25, 26]. That is, the alkaline form of native horseradish peroxidase has smaller magnetic susceptibility value (/~eff = 2.66fl) than that of metmyoglobin (~er~ z 5.04fl) and its absorption spectrum resembles that of a low spin type. In spite of a diversity of proposals [27-32], no established result has been offered concerning the kind of ligands existing above and below the heme iron. In this report MCD spectra of native horseradish peroxidase from the acid form (pH < 7) to a peculiar alkaline form (pH > 12) were finely measured and attempts were made to deduce the 5th and the 6th ligands by examining changes in electronic states. EXPERIMENTAL PROCEDURES Horseradish peroxidase (Sigma type II) and myoglobin (Sigma type II) were purchased and used without further purification. The purity defined as the absorptivity ratio at 403 nm and 275 nm, was 1.48 for the horseradish peroxidase. Cytochrome c (Saccharomyces oviformis) was supplied from Sankyo Pharmaceutical Co. Ltd. Absorption spectra were measured at 0 °C in a Hitachi EPS-3T spectrophotometer, using cuvettes of 10mm (700-1800nm), 5 mm (450-700nm) and 1 mm (250-450 nm) optical pathes. MCD spectra were recorded on a JASCO J-20A (2501000 nm) and a JASCO J-200 (1000-1800nm) spectropolarimeter equipped with JASCO electromagnets to produce longitudinal magnetic fields up to 15 000 gauss at samples. Magnetic field was determined with freshly prepared ferricyanide using the established value; [0]M (422nm) 1.0 [1]. The magnet was operated in a normal mode producing the longitudinal magnetic field parallel to the direction of the light. To separate natural circular dichroism (CD) from each MCD spectrum, the spectra were recorded twice; once with magnetic field parallel and then without any magnetic field. MCD magnitude was expressed by the molar ellipticity per gauss ([0]M; degree cm2/dmol gauss) on the basis of the heme concentration. Samples were prepared in a 0.1 M phosphate buffer (pH 7.01), then pH was adjusted to the desired pH values by adding concentrated sodium hydroxide or hydrochloric acid solution. A cell of 5 mm (450-700 nm) or 1 mm (250-450 nm) path and concentration of 2 to 4/zM were used for measurements in the near ultraviolet and visible region; the cell was placed in a quartz Dewar vessel containing ice-water. For measurements in the near infrared region a 10 mm cell was used at concentrations of 0.4 to 0.9 mM in a 0.15 M deuterium oxide phosphate buffer at ambient temperatures. An automatic slit width affording spectral band width of less than 2 nm was found sufficient to resolve all bands. Scan rate of 5 or 10 nm/min was used with a time constant of 4 s. =
RESULTS Fig. 1 shows the MCD and absorption spectra of native horseradish peroxidase. The MCD and absorption spectra remained unchanged in the pH range from 5.20 to 9.00. The MCD spectra in this pH range are characteristic of ferric high spin hemoproteins. The visible MCD spectra show two sinusoidal curves; the larger one consists of a trough at 550-551 nm and a peak at 524 nm with a crossover at
342
........ pH 5.20 2
- - I 3117.01 406
!
299
-474
- - - p H 9.00
^
0 E
550"I -2 -3
424
1.0
4O2
-4
~
-5
x
496
300
&O0
500
35
600
uJ
700
~(nm)
Fig. 1. MCD and absorption spectra of native (Fe(IlI)) horseradish peroxidase in an acid form. Concentration, 1.924.10 -~ M in 0.1 M potassium phosphate buffer (pH 7.01); path length 1 mm below 450 nm, 5 mm above 450 nm; magnetic field 11 400 gauss; temperature 0 °C. Vertical line = change of e scale as indicated by magnification factor. 535 nm, based mainly on the Q absorption band, and the smaller one has a trough at 658 nm and a peak at 615 nm with a crossover at 626 nm associated mainly with charge-transfer bands [1, 28]. A larger apparent A term in the Soret region is composed of a trough at 424 nm and a peak at 406 nm with a crossover at 414 nm. Fig. 2 illustrates the Soret M C D spectra at various p H values corresponding to the M C D changes associated with the transition from acid to alkaline forms. The 424 nm trough developed its intensity with an increase of p H to [0]M of --22.4 at p H 12.01. The peak at 406 nm of the two peaks in the Soret region was predominant in th¢ higher p H region (pH > 11.10) resulting in the M C D peak with [0]M = 16.4 at p H 12.01. The intensity of the Soret trough of the alkaline form ([0]M = --22.4) for the native horseradish peroxidase is far bigger than that observed for metmyoglobin hydroxide ([0]M = --8.3) which is evidence that the alkaline form of the native horseradish peroxidase is composed of more low-spin species than that of metmyoglobin. This is consistent with the result obtained from magnetic susceptibility and absorption spectra [25, 26]. The magnitudes of the Soret M C D spectra at the positive and negative extrema are plotted as a function of p H in Fig. 3. Both the peak at 406 nm and the trough near 424 nm increased throughout the p H range measured, indicating a conversion from high to low spin complex. The midpoint for the transition observed occurred at p H 11.0, which is in good agreement with the results previously reported [25-29, 32]. Assuming that M C D recorded at p H 7 corresponds to the 100% acid form and
343
pH 12.01 ,pH 11.30 ,pH 11.10 ,pH 10.80 ,pH 10.50 .pH 10.25 _DH 10.00
10
0
-I0
-20
MCD at v a r i o u s pHs
400 /~(nrn )
450
Fig. 2. Soret MCD spectra of Fe(III) horseradish peroxidase corresponding to the transition from acid to alkaline forms. Concentration, 2.28.10 -4 M in water; path length 1 m m ; magnetic field 11 400 gauss; temperature 0 °C. ^.alkaline -form acid form
.~,~. ~ , (,n
--
O
~
Sorer 0
[e]m
Peak co
c~
p. . . .
r,D
'o
"0 -r
tog acid form Soret Trough [O]M alkaline form Fig. 3. M C D spectrophotometric titration of horseradish peroxidase. The data are taken from Fig. 2.
344
403
- - Nb(lll')-H20
295
- - - ; ge(X) Protoporphyrin IX Dimethyl Ester CHsCO0-
2
,r,\,
/'l
I I
567
~o 517
-I
64~ -2 -3 -4 ;21 -5
~
503 502
633
300
400
500
600
x
700
A (nm)
Fig. 4. M C D and absorption spectra of metmyoglobin acid form ( p H 7.01) and the metmyoglobin carboxylate complex. Concentration, 2.81.10-4 M for both derivatives; path length 1 mm below 450 nm, 5 mm above 450 nm; magnetic field 11 400 gauss; temperature 0 °C. M C D of acetato-iron (III) p r o t o p o r p h y r i n IX dimethyl ester was replotted from data of ref. 33.
that formed at pH greater than 12 to the 100K alkaline form, we plotted the ratio of the acid to the alkaline form as a function of pH in Fig. 3. The pK value of 11.0 is calculated from either extremum for the transformation of acid to alkaline form. No slope of the data from pH 5 to 9 in Fig. 3 might reflect the absence of any intermediate complexes throughout this region at 0 °C. Fig. 4 shows the MCD and absorption spectra of metmyoglobin carboxylate together with those of the acid metmyoglobin [1] and a carboxylate complex of Fe(III) protoporphyrin IX dimethyl ester [33] from published data. The acid form of horseradish peroxidase gave an MCD spectrum somehow different from that of acid metmyoglobin. The visible MCD spectrum of metmyoglobin carboxylate is strikingly similar in shape and magnitude to that of acid metmyoglobin.
345
Figs. 5 and 6 illustrate the MCD and absorption spectra in the near infrared region for acid and alkaline forms of native horseradish peroxidase, and a cyanide complex of native horseradish peroxidase, ferric cytochrome c at p2H 6.86 and 11.90, I
I
I
,,,
i ,, ..},,,
I
i f HRP -;p2H 6.86
',,,
,,"
,,,
]/]//"
...... ;p2H12.10
'\
"
o x
ol
700
!_
I
8(30
I
900
I
1000
I
II00 1200
.... * .....
I
1300
1400 1500
k(nm)
Fig. 5. M C D and absorption spectra of Fe(III) horseradish peroxidase ( ) and its alkaline form (. . . . . ) in the near infrared region. Concentration 4.64.10 -+ M in 1/15 M deuterium oxide phosphate buffer (p2H 6.86) for native horseradish peroxidase and 3.74' 10 -4 M in deuterium oxide (p2H 12.01) for the alkaline form; path length 10 ram, magnetic field 14 700 gauss; temperature 20 °C. The bars indicate the average noise levels. I
I
I
I
1
I
I
I
I
I
; HRP(]]])-CN.........: Cyt,c( p~H6.86) - . - ; Cyt,c(pRll.90)
/'"t~"~-"~
....
"+
-
I
3
-
~
-
MCD
2o x
\ .L 6 ~4 ~J
2 I
)0
I~"~'1
900
I
II00
I
I
1300 ~.(nm )
I
I
1500
I
I
1700
"7
1900
Fig. 6. M C D and absorption spectra of Fe(IIl) horseradish peroxidase cyanide complex (-), cytochrome c (pZH 6.86) ( . . . . . . ), cytochrome c (p2H 11.90) ( - - - - - ) , metmyoglobin imidazole (. . . . ) in the near infrared region. Concentrations; 3.98.10 -+, 8.60.10 -4, 2.00.10 -3 M in 1/15 M deuterium oxide phosphate buffer (p2H 6.86) for native horseradish peroxidase cyanide complex, cytochrome c (p2H 6.86), metmyoglobin imidazole, respectively, 8.60.10 -+ M for cytochrome c alkaline form in deuterium oxide (p2H 11.90). The cyanide and imidazole complexes were obtained with addition of excess solid potassium cyanide and imidazole, respectively. Path length 10 mm except for metmyoglobin imidazole (2 mm); magnetic field 14 700 gauss except for metmyoglobin imidazole (11 400 gauss); temperature 20 °C, the bars indicate the average noise levels. MCD of metmyoglobin imidazole was replotted from data of ref. 10.
346 [
I
i ~ l i ~ r f l l r
I
5 1 I l l 1
2
-1 -2 --3
-
Cytochrom¢C pH 11.0
---
,,
~
// f
4
---o---HRP Alkatin¢ form pill2,0
/"" i /
/,.
--4
V I
,
~
450
~
J
I
500
t
~
J
I
I
550 3,(nm)
i
L
I
600
~
L
~
f
I
650
Fig. 7. MCD spectra of cytochromec (pH 11.0and 7.0), cytochromeb5and horseradish peroxidasealkaline form (pH 12.0). The data of cytochromes are replotted from data of ref. 15. and metmyoglobin imidazole. The acid form of horseradish peroxidase exhibited troughs at 890 and 1270 nm and peaks at 760 and 1060 nm being somehow similar to that of the acid metmyoglobin [10]. The alkaline form of native horseradish peroxidase showed a peak at 1100 nm with a shoulder on the shorter wavelength side. Fig. 7 demonstrates the visible MCD spectrum for the alkaline form of horseradish peroxidase together with those for some other hemoproteins as low spin models, i.e., cytochrome c at pH 7.0 and 11.0, and cytochrome bs from published data [15]. All these MCD spectra show "S shaped" bands around 540-600 nm, and bell shaped positive bands from 460-530 nm which have been ascribed to the Q and charge-transfer bands, respectively [1]. These charge-transfer bands could not be resolved by electronic absorption spectra, but were clearly detected by MCD spectra. These bands have been assigned to the transition from porphyrin b2u and a'2u er orbitals to iron dn (eg) orbitals [1, 10]. DISCUSSION
The acid form of native horseradish peroxidase exhibited on the whole the MCD spectra peculiar to high spin hemoproteins. Some complex features in the lower wavelength side of the Soret band were discussed in the previous paper [23]. Two different types of visible MCD patterns have been observed among high spin hemoproteins with a histidine as the 5th ligand: one is acid metmyoglobin and metmyoglobin carboxylate, and the other is metmyoglobin fluoride and the high spin component of metmyoglobin hydroxide. This difference has been ascribed to the nature of the 6th ligand of metmyoglobin derivatives [1]. The MCD spectra in Fig. 1 indicate that the acid form of native horseradish peroxidase can be grouped into the high spin hemoproteins of the acid metmyoglobin type. This is consistent with implications from photometric [34, 35] and ESR studies [36] that the proximal ligand is a nitrogen-containing group, probably histidine. However, there exist some fine differences in the intensity ratio of the sinusoidal MCD spectra around 540 nm to
347 that of 640 nm between the acid form of native horseradish peroxidase ( ~ 2) and the acid metmyoglobin ( ~ 1/2). As to this intensity ratio the carboxylate complex of Fe(III) protoporphyrin IX dimethylester in Fig. 4 exhibited a similar ratio ( ~ 2) to that of the acid form of horseradish peroxidase [33]. Since this complex has a pentacoordinated heme, the similarity in the intensity ratio might suggest an electronic structure of the iron for the acid form of native horseradish peroxidase similar to that formed in the pentacoordinated heme. This may be consistent with proton magnetic resonance evidence which suggests the absence of iron-coordinated water in horseradish peroxidase [30]. (However, it should be added that a different interpretation has been proposed for the proton magnetic resonance result [31]). On the other hand, the resonance Raman spectrum of the acid form of native horseradish peroxidase was reported to be different from acid methemoglobin, and this was interpreted as a result of less doming of the porphyrin ring for the former relative to the latter [37]. Therefore the difference in the MCD intensity ratio of the 540 to 640 nm band between the acid form of native horseradish peroxidase and acid metmyoglobin may relate to these structural differences of the porphyrin ring. The visible MCD spectra especially for ferric high spin derivatives are very sensitive to the electronic structures of both porphyrin and iron, because it depends on the complex configuration interactions between the porphyrin Q state and the charge-transfer state associated with iron dn (eg) orbitals. The electronic states of the heine iron depend sensitively on the position of the iron in the porphyrin ring (i.e., doming structure). This position may be determined by the ligand field strength and balance of the apical ligands, and protein structures which control the position and ligand field of the apical ligand atoms. Hence the similarity in the MCD intensity ratio between the acid form of horseradish peroxidase and acetato-iron(III) protoporphyrin IX dimethyl ester may suggest that similar electronic states of the heme iron are formed between the two by the similarity in the iron positions. In the near infrared region the acid form of native horseradish peroxidase exhibited the MCD spectrum which can be grouped into the high spin type of acid metmyoglobin instead of that of metmyoglobin fluoride. However the acid form of native horseradish peroxidase gave an MCD inflection point at 1200 nm in longer wavelength by 100 nm than that of the acid metmyoglobin [10]. The relative magnitudes of the peak to the trough were also reversed between them. These results suggest that, though the acid horseradish peroxidase has a fairly similar ligand state to that of acid metmyoglobin, there exist some fine differences between them. These are consistent with the discussion from the MCD spectra in the Soret and visible regions. Structures of the alkaline form of native horseradish peroxidase have attracted many investigators. It has been established at least that the alkaline form of native horseradish peroxidase belongs to low spin hemoproteins [25, 26, 38]. This is clearly different from the fact that the alkaline form of metmyoglobin has more fraction of high spin component ( ~ 65 ~) than that of low spin component ( ~ 35 ~) at ambient temperatures [28, 39]. Many ideas have been proposed as to the kind of axial ligands of the alkaline form of native horseradish peroxidase [27-32]. The MCD spectral results are consistent with those previously reported in that it clearly displayed the MCD spectra of hemoproteins containing mainly low spin heine chromophores. Furthermore it furnished some evidence to determine the kinds of axial ligands as described below.
348 The MCD magnitude of the Soret band for the ferric hemoproteins has been established to relate closely to the low spin components present [1]. Since the MCD magnitude ([0]~) depends on both the magnetic moment and the electric transition moment of the molecules or ions, the value ([0]M/emM, where emM is the millimolar extinction coefficient), which depends essentially on the magnetic moment, has been proposed to be useful as a spectral index of the MCD spectra [40-43]. Hence the magnitudes of the peak to trough in the Soret MCD bands divided by the millimolar extinction coefficients of the Soret peaks were calculated to be 0.44, 0.34, 0.31, 0.49 and 0.70 for the alkaline form of native horseradish peroxidase, ferric cytochrome bs, metmyoglobin imidazole, and ferric cytochrome c at pH 6.4 and that at pH 11.5, respectively. Ferric cytochrome b5 and metmyoglobin imidazole are the reference hemoproteins with two histidinyl imidazoles as axial ligands. Ferric cytochrome c at pH 6.4 and that at pH 11.5 are the models for the hemoproteins having histidinyl imidazole and methionyl sulfur, and histidinyl imidazole and lysinyl nitrogen, respectively. The calculated values indicate that the axial ligands of the alkaline form of horseradish peroxidase are different from that of ferric cytochrome c at pH 11.0, i.e., the pair of histidinyl imidazole and lysinyl nitrogen. It shows fairly a close value to that of ferric cytochrome c at pH 6.4, but it is not so different from that of ferric cytochrome bs or metmyoglobin imidazole. The visible MCD patterns shown in Fig. 7 also gave a similar conclusion. Fig. 7 shows that the MCD spectra of the alkaline form of native horseradish peroxidase resemble in shape to that of ferric cytochrome c at pH 6.4, but a band shift by ~ 15 nm is noticeable. Though it somehow resembles in shape and magnitude to that of ferric cytochrome bs, a band shift is also noticeable. Furthermore, contrary to the bis-imidazole type complex whose MCD peak at Q0-0 band (around 560 nm) surpasses the charge-transfer bands (470-530 nm) [1] in magnitude, the alkaline form of native horseradish peroxidase maintains the same MCD intensity in these two regions. The alkaline form gives somehow different MCD spectra from that of ferric cytochrome c at pH 11.0 in both shape and band position. So far no definite conclusion can be drawn from the MCD spectra in the Soret and visible regions. However the near infrared MCD spectra gave further implication to determine the axial ligands of the alkaline form of native horseradish peroxidase. As we have discussed in detail for metmyoglobin derivatives in the previous paper [10], the MCD observed in the near infrared region especially for ferric low or high spin derivatives is due to charge-transfer transitions from the porphyrin occupied alu and a2u 7r to iron d~ (e~) orbitals [10, 28]. The alkaline form of native horseradish peroxidase, metmyoglobin imidazole, and ferric cytochrome c at p2H 6.86 and that at pZH 11.90 exhibited similar positive MCD peaks associated with the absorption peaks in the near infrared region. These positive MCD bands have been observed for other ferric low spin derivatives such as metmyoglobin cyanide, the cyanide complex of native horseradish peroxidase (Fig. 6), metmyoglobin imidazole, metmyoglobin azide, and the low spin component of metmyoglobin hydroxide [10]. Although all ferric low spin derivatives displayed similar MCD patterns, there exist substantial differences in the absorption wavelengths. Though the absorption wavelength depends on many factors because these bands are charge-transfer bands mentioned above [10, 28], the overall positions of the absorption bands seem to depend on the kind of axial ligands. It is noticed that the band positions of the near
349 infrared MCD spectra show correlation to the so-called spectro-chemical series. Thus, the MCD peak of the cyanide derivative exists at the longest wavelength (1530 nm), those of imidazole (1520nm), azide (1230nm) and the low spin component of hydroxide complexes (990 nm) following in this order from the longer wavelength. It is very notable that the alkaline form of native horseradish peroxidase exhibited the near infrared MCD peak at 1100 nm was completely different from those of metmyoglobin imidazole (1520 nm), ferric cytochrome c at p2H 6.86 (1540 nm) and ferric cytochrome c at p2H 11.90 (1520 nm). Hence the near infrared MCD spectra indicate that the axial ligands of the alkaline form are different from the pairs of histidine and histidine, histidine and methionine, and histidine and lysine. Among the ferric low spin derivatives studied, the low spin component of metmyoglobin hydroxide showed the near infrared MCD peak at the closest wavelength (990 nm) to that of the alkaline form of native horseradish peroxidase (1100 nm), though it still shows the MCD peak at shorter wavelength by 110 nm. Since, as described above, the near infrared bands shift to a longer wavelength as the ligand field of apical ligands increases, a histidine and a hydroxide ion with some interactions to strengthen the ligand field seem to be a possible pair of apical ligands of the alkaline form. A similar structure has been proposed for the alkaline form of horseradish peroxidase by Yamazaki et al. [27] i.e., the heme vicinity with a hydroxyl group whose basicity at the oxygen is strengthened by some reasons possibly by a hydrogen bonding with the distal histidine. Naturally this is, of course, a possibility. However, this does not exclude other possibilities, such as a pair of a histidine and some other ligand with overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide whose near infrared MCD peaks exist at 990 and 1230 nm, respectively. It may be mentioned here that the alkaline form of horseradish peroxidase exhibited clearly different MCD spectra in the Soret and visible regions, irrespective of the similarity in the near infrared region just mentioned. We need to stress here the fact that the alkaline form resembles the low spin component of metmyoglobin hydroxide. As mentioned earlier, metmyoglobin hydroxide is composed of about 65 ~ high spin and 35 ~o low spin components in thermal equilibrium at ambient temperatures. Although, in the Soret and visible regions, the contribution of the high spin component greatly affects the observed MCD spectra, if we extract the MCD component of the low spin fraction, it will be found that the similarity between the alkaline form and the low spin component of metmyoglobin hydroxide is still held in the Soret and visible regions. In conclusion the acid form of horseradish peroxidase has on the whole a similar electronic structure to that of acid metmyoglobin. However, fine differences between them were ascribed to the differences in the electronic states of the heme iron derived from structure of the heme unit, such as doming of the porphyrin ring induced by strength and balance of the apical ligands associated with protein structures. The alkaline form of native horseradish peroxidase exhibited somehow different MCD spectra from those of typical low spin hemoproteins. The near infrared MCD spectra implicated that the alkaline form seems to have a pair of axial ligands possessing a histidine and another ligand with overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.
350 ACKNOWLEDGEMENTS The authors would like to express their t h a n k s to Professor Isao Y a m a z a k i of H o k k a i d o University for valuable discussions. The authors also wish to t h a n k the Mitsubishi Scientific F o u n d a t i o n for the financial support to construct the near infrared C D spectropolarimeter.
REFERENCES 1 Vickery, L. E., Nozawa, T. and Sauer, K. (1976) J. Am. Chem. Soc. 98, 343-350 2 Schooley, D. A., Bunnenberg, E. and Djerassi, C. (1965) Proc. Natl. Acad. Sci. U.S. 53,579-589 3 Dratz, E. A. (1966) Ph.D. Thesis, University of California, Radiation Laboratory Report UCRL17200 4 Kobayashi, H., Shimizu, M. and Fujita, I. (1970) Bull. Chem. Soc. Japan 43, 2335-2341 5 Djerassi, C., Bunnenberg, E. and Elder, D. L. (1971) Pure Appl. Chem. 25, 57-90 6 Garnier, A., Bolard, J. and Danon, J. (1972) Chem. Phys. Lett. 15, 141-143 7 Bolard, J. and Gamier, A. (1972) Biochim. Biophys. Acta 263, 535-549 8 Yoshida, S., Iizuka, S., Nozawa, T. and Hatano, M. (1975) Biochim. Biophys. Acta 405, 122-135 9 Cheng, J. C., Osborne, G. A., Stephens, P. J. and Eaton, W. A. (1973) Nature 241,193-194 10 Nozawa, T., Yamamoto, T. and Hatano, M. (1976) Biochim. Biophys. Acta 427, 28-37 11 Greenwood, C. and Wilson, M. T. (1971) Eur. J. Biochem. 22, 5-10 12 Sutherland, J. C. and Klein, M. P. (1972) J. Chem. Phys. 57, 76-86 13 Risler, J. L. and Groundinsky, O. (1973) Eur. J. Biochem. 35, 201-205 14 Briat, B., Berger, D. and Leliboux, M. (1972) J. Chem. Phys. 57, 5606-5607 15 Vickery, L. E., Nozawa, T. and Sauer, K. (1976) J. Am. Chem. Soc. 98, 351-357 16 Dolinger, P. M., Kielczewski, M., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E. and Djerassi, C. (1974) Proc. Natl. Acad. Sci. U.S. 71,399-403 17 Dawson, J. H., Dolinger, P. M., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E. and Djerassi, C. (t974) Proc. Natl. Acad. Sci. U.S. 71, 4594-4597 18 Vickery, L., Salmon, A. and Sauer, K. (1975) Biochim. Biophys. Acta 386, 87-98 19 Shimizu, T., Nozawa, T., Hatano, M., Imai, Y. and Sato, R. (1975) Biochemistry 14, 4172-4178 20 Palmer, G., Babcock, G. T. and Vickery, L. E. (1976) Proc. Natl. Acad. Sci. U.S 73, 2206-2210 21 Babcock, G. T., Vickery, L. E. and Palmer, G. (1976) J. Biol. Chem. 251, 7907-7917 22 Thomson, A. J., Brittain, T., Greenwood, C. and Springall, J. (1976) FEBS Lett. 67, 94-98 23 Stillman, M. J., Hollebone, B. R. and Stillman, J. S. (1976) Biochem. Biophys. Res. Commun. 72, 554-559 24 Nozawa, T., Kobayashi, N. and Hatano, M. (1976) Biochim. Biophys. Acta 427, 652-662 25 Keilin, D. and Hartree, E. F. (1951) Biochem. J. 49, 88-104 26 Theorell, H. and Ehrenberg, A. (1951) Acta Chem. Scad. 5, 823-848 27 Yamazaki, I., Hayashi, Y., Makino, R. and Yamada, H. (1975) "Proceedings of the Japan and U.S.A. Seminar, Hawaii", in the press 28 Smith, D. W. and Williams, R. J. P. (1970) Structure and Bonding 7, 1-45 29 Williams, R. J. P., Wright, P. E., Mazza, G. and Ricard, J. R. (1975) Biochim. Biophys. Acta 412, 127-147 30 Lanir, A., and Schejter, A. (1975) Biochem. Biophys. Res. Commun. 62, 199-203 31 Vuk-Pavlovic, S. and Benko, B. (1975) Biochem. Biophys. Res. Commun. 66, 1154-1159 32 Iizuka, T., Ogawa, S., Inubushi, T., Yonezawa, T. and Morishima, I. (1976) FEBS Lett. 64, 156158 33 Dawson, J. H., Holm, R. H., Trudell, J. R., Barth, G., Linder, R. E., Bunnenberg, E., Djerassi, C. and Tang, S. C. (1976) J. Am. Chem. Soc. 98, 3707-3708 34 Brill, A. S. and Sandberg, H. E. (1968) Biophys. J. 8, 669-690 35 Brill, A. S. and Sandberg, H. E. (1968) Biochemistry 7, 4254-4260 36 Yonetani, T., Yamamoto, H., Erman, J. E., Leigh, J. S. and Reed, G. H. (1972) J. Biol. Chem. 247, 2447-2455 37 Gopa, R. and Spiro, T. S. (1974) Biochemistry 13~ 5317-5323
351 38 39 40 41 42 43
Tamura, M. (1971) Biochim. Biophys. Acta 243, 249-258 Iizuka, T., and Kotani, M. (1969) Biochim. Biophys. Acta 181, 275-286 Buckingham, A. D. a/ad Stephens, P. J. (1966) Annu. Rev. Phys. Chem. 17, 399--432 Schatz, P. N. and McCaffery, A. J. (1969) Q. Rev. Chem. Soc. 23, 552-584 Kaito, A., Tajiri, A. and Hatano, M. (1975) J. Am. Chem. Soc. 97, 5059-5062 Kaito, A., Tajiri, A. and Hatano, M. (1976) J. Am. Chem. Soc. 98, 384-388
