Resonance raman spectroscopic characterization of the heme coordination and spin state in the alkaline form of horseradis peroxidase

Biochimica et Biophysica A cta, 789 (1984) 80- 86 Elsevier

80

BBA 31971

R E S O N A N C E RAMAN S P E C T R O S C O P I C C H A R A C T E R I Z A T I O N O F T H E H E M E C O O R D I N A T I O N AND S P I N S T A T E IN T H E ALKALINE F O R M OF H O R S E R A D I S H PEROXIDASE JAMES TERNER

*

and DAVID E. REED

Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284 (U.S.A.) (Received February 6ttr, 1984) (Revised manuscript received May 17th, 1984)

Key words: Heine protein," Resonance Raman," Enzyme intermediate," Horseradish peroxidase," Heine co-ordination; Alkaline transition

Resonance Raman spectra of the alkaline forms of horseradish peroxidase isoenzymes are indicative of six-coordinate low-spin hemes. Comparisons are made with resonance Raman spectra of horseradish peroxidase Compounds II and X. The v4 porphyrin frequencies of the Fe(IV) derivatives, that are used as oxidation state marker bands, have been found to be not significantly higher than those of the low-spin Fe(IIl) hemes of the alkaline forms.

Introduction

Peroxidase as isolated from horseradish is a heme protein that contains protoporphyrin IX at the active site, similar to hemoglobin, myoglobin, catalase and cytochrome P-450 [1]. The coordination number of the horseradish peroxidase iron has historically been a topic of discussion. Horseradish peroxidase had, until recently, been believed to contain a six-coordinate heme with a water molecule in the sixth coordination position [1]. At present, spectroscopic evidence strongly supports penta-coordination [2,3] in the neutral resting enzyme. Horseradish peroxidase will, however, readily assume six-coordinate heme configurations. One of the most easily obtainable six-coordinate forms is the alkaline form. Reversible transformations between the neutral and alkaline forms of horseradish peroxidase are obtained by adjustment of the pH. Alkaline transitions of heme proteins such as cytochrome c [4], hemoglobin [5], * To whom correspondence should be addressed. 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

myoglobin [5] and horseradish peroxidase have been well documented. The alkaline transition in horseradish peroxidase is plainly evident by a color change from brown to red with concomitant changes in the optical absorption spectrum, and a switch from a high- to low-spin iron [5-7]. The resonance R a m a n spectra presented in this paper clearly show a transition from a five-coordinate high-spin heine at neutral pH, to a six-coordinate low-spin configuration at alkaline pH. The physiological function for the alkaline transition of peroxidases is not understood. Chance [8] has reported that the alkaline form of horseradish peroxidase does not react with peroxides. The alkaline transition points of various peroxidases vary. Horseradish peroxidase isoenzymes B and C have a p K of 10.7 [9] while isoenzyme A-1 has a p K of 9.2 [10]. Isoenzyme P-7 of turnip peroxidase has a transition point even closer to neutrality, with a pK of 8.4 [11]. Because of alkaline transitions close to neutral pH, it has been thought that a physiological function is likely. In this paper we compare resonance Raman

81 spectra of various high-spin six-coordiante forms of horseradish peroxidase isoenzymes with those of the alkaline form and the neutral resting enzyme. Materials and Methods

Horseradish peroxidase was isolated from horseradish roots by a m m o n i u m sulfate fractionation (Schwarz-Mann enzyme grade (NH4)2SO4), and DEAE- and CM-cellulose ion-exchange chromatography (Whatman DE-52 and CM-52) following the procedures of Shannon et al. [12]. Several repetitions of the ion-exchange chromatographic steps were sometimes needed to remove fluorescent impurities which tended to obscure the resonance R a m a n spectra under visible laser excitation frequencies. Samples of isoenzyme A-1 were studied separately from a mixture of isoenzymes B and C. Diafiltrations and concentrations were performed with ultrafiltration cells (Amicon) fitted with PM-10 membranes. Horseradish peroxidase compound II [1] was prepared by rapidly mixing equimolar quantities of horseradish peroxidase and dilute H202. Compound X was prepared following the procedure of Shahangian and Hager [13]. Samples were placed in a spinning cell or a recirculating jet apparatus [14] for R a m a n measurements. The resonance R a m a n excitation source was a krypton ion laser (Spectra-Physics model 171) equipped with an ultra-high-field magnet. Samples were excited transversely at low (less than 5 mW) laser power. Scattered light was collected at 90 ° by a Canon f/1.2 50 m m lens and focused onto the slit of a 0.5 m spectrograph (Spex model 1870) equipped with interchangeable 600, 1200, and 1800 g r o o v e / m m gratings. Glass filters (Coming or Schott) were used when necessary to attenuate scattered laser light. The detection system was an optical multichannel analyzer (Princeton Applied Research Corp. Model 1215/1216) with a Model 1224 silicon-intensified vidicon detector head. The laser wavelengths used were 406.7, 413.1, and 482.5 nm to observe Soret enhancement and 530.8 nm to observe a-fl enhancement [15]. The resonance Raman spectra were calibrated with the known frequencies of indene [16]. The resolution was 2 c m - 1 and band frequences are accurate to + 1 cm-1 for strong isolated bands. The band assignments and

numbering system follow those given by Abe et al. [17] and Choi et al. [18,19] for the modes of nickel octaethylporphyrin. The bands are located by the frequency patterns and band polarizations which are either polarized (p), depolarized (dp), or anomalously polarized (ap) [15]. Resonance Raman spectra of neutral horseradish peroxidase, alkaline horseradish peroxidase, and horseradish peroxidase compound II are compared in Fig. 1 with Soret excitation in which the polarized bands predominate; and in Fig. 2 with a-fl excitation, in which depolarized and anomalously polarized bands predominant. A listing of frequencies, assignments and observed polarizations are given in Table I. Certain frequencies listed in Table I and the figures were obtained by deconvolution of complex bands (such as the complex band at 1630 cm 1, seen under Soret enhancement) through polarization measurements. No smoothing was performed on any of the resonance R a m a n spectra shown in this paper. Results

From the resonance Raman spectra of Figs. 1 and 2 it is most apparent that the horseradish peroxidase heme coordination and spin state has changed from a five-coordinate high-spin heme at neutral p H to a six-coordinate low-spin heme at high pH. Spin marker [15] bands, vl0, v19, vz, vll and v3 appear at 1631 (dp), 1574 (ap), 1572 (p), 1549 (dp), 1491 and 1505 (both p, these two bands appear to be v3, which has been split; see Discussion) cm-1 for horseradish peroxidase isoenzyme A-1 at neutral p H (Figs. l a and 2a). These frequencies have been interpreted as being indicative of a five-coordinate high-spin heme by comparison with five-coordinate model hemes [3]. At alkaline pH, spin marker frequencies vl0, v19, v2, v~, and v3 shift to 1637 (alp), 1586 (ap), 1584 (p), 1558 (dp), and 1505 (p) cm -1 (Figs. lc and 2c). These frequencies are similar to those in the resonance R a m a n spectra of oxyhemoglobin, ferricytochrome c, ferrihemoglobin cyanide and ferrihorseradish peroxidase cyanide which all contain six-coordinate low-spin iron(III) hemes [20]. Isoenzymes B and C show a similar transformation from neutral (Figs. l b and 2b) to alkaline p H (Fig. lc). (Isoenzymes B and C undergo some degradation at

82 Z

ro

? °ll~

/

nI-IHRPA-I

~#

(a)

0,

(~

~--~

-~' ~o

~

~

~/~ o ~,~ ~A~O

~~M~~--

HRP A-I

pH7

~o

m CO tO

(b)

c~ --

~

ro -~

~ 0 I I~ - ~ ~- m

V

r,o~ ----

~.

HRPB,C

B,C

(cl

_-~

(c)

_~

--

,~

~-I

HRP~-~

(d)

m(e)

(f)

~I

m

~_~ __.~oT ~--

~ '~"

- -

~ ~

/I---/~-*~

T

o~

Fig. 1. Resonance Raman spectra of horseradish peroxidase (HRP), 0.1 mM, using 406.7 nm excitation. (a) isoenzyme A-1 (pH 7.0), 0.02 M Na2HPO4-NaH2PO4; (b) Isoenzymes B and C (pH 7.0), 0.02 M Na2HPO4-NaH:PO4; (c) isoenzyme A-1 (pH 10,0), 0.02 M NazCO3-NaHCO3; (d) isoenzymes B and C (pH 11.5), 0.025 M glycine; (el compound II, isoenzymes B and C (pH 7.0), 0.02 M Na2HPO4-NaH2PO4 with 2 molar equivalents of diluted hydrogen peroxide; (f) compound X, isoenzymes B and C (pH 10.7), 0.005 M Na2CO3-NaHCO 3 and 0.1 mM sodium chlorite.

a l k a l i n e p H , p a s t their a l k a l i n e t r a n s i t i o n p o i n t , c a u s i n g a f l u o r e s c e n t i n t e r f e r e n c e w h i c h results in l o w - q u a l i t y r e s o n a n c e R a m a n s p e c t r a w h e n excited u n d e r a-/3 excitation. T h i s p r o b l e m was n o t o b s e r v e d for i s o e n z y m e A - l , b e c a u s e the a l k a l i n e t r a n s i t i o n p o i n t is closer to n e u t r a l i t y . ) It is i n t e r e s t i n g to n o t e that while r e s o n a n c e R a m a n s p e c t r a of the two sets of i s o e n z y m e s are slightly d i s s i m i l a r at n e u t r a l p H (Figs. l a a n d l b ) , the c o r r e s p o n d e n c e is m u c h closer at a l k a l i n e p H (Figs. l c a n d

Fig. 2. Resonance Raman spectra of horseradish peroxidase (HRP), 1 raM, using 530.9 nm excitation. (a) isoenzyme A-1 (pH 7), 0.02 M NazHPO4-NaHzPO4. (b) isoenzymes B and C (pH 7), 0.02 M Na2HPO4-NaH2PO4. (c) isoenzyme A-1 (pH 9.2), 0.005 M Tris-acetic acid, 0.01 M NaCI. (d) compound II, isoenzymes B and C (pH 7), 0.02 M Na2HPO4-NaHePO 4. l d ) , suggesting, t h o u g h c e r t a i n l y n o t p r o v i n g , the p o s s i b i l i t y of the s a m e sixth l i g a n d in b o t h cases. T h e r e s o n a n c e R a m a n s p e c t r a of a l k a l i n e h o r s e r a d i s h p e r o x i d a s e (Figs. lc, d a n d 2c) are very similar to the r e s o n a n c e R a m a n s p e c t r u m of h o r s e r a d i s h p e r o x i d a s e c o m p o u n d II (Figs. l e a n d 2d) a n d c o m p o u n d X (Fig. lf). A n i n t e r e s t i n g s i m i l a r i t y is the close c o r r e s p o n d e n c e in f r e q u e n c y of 1'4. This f r e q u e n c y is k n o w n to be i n d i c a t i v e of the o x i d a t i o n state of the h e m e iron, with i r o n ( I I ) at 1360 c m l, i r o n (III) at 1373 c m - 1 a n d i r o n ( I V ) at 1381 c m -1 [15]. W h i l e the v a l u e of l,4 is k n o w n to vary s m o o t h l y in F e ( I I ) h e m e s with the increasing ~r-acceptor a b i l i t y of the axial l i g a n d s [21,22], this g e n e r a l i z a t i o n is n o t a p p l i c a b l e to F e ( I I l ) heroes [15]. I n Figs. 1 a n d 2, u4 is seen to u n d e r g o a shift f r o m 1373 c m -~ at p H 7, to 1378 or 1379 c m ~ at a l k a l i n e p H . This is in c o m p a r i s o n to a u4 v a l u e of 1376 c m ~ that we o b s e r v e for horsera-

83 TABLE I M O D E ASSIGNMENTS, OBSERVED POLARIZATIONS A N D FREQUENCIES IN cm -1 FOR THE RESONANCE RAMAr~ SPECTRA SHOWN IN FIGS. 1 AND 2

The mode assignments follow Refs. 17-19. HRP denotes horseradish peroxidase. Mode

Observed polarization

Ulo Big uc= c (2) vinyl Vc=c (1) vinyl P37 Eu /'19 A2g u2 Alg ~'11 Blg V3s E u

dp p p P ap p dp p

HRP A-1 (pH 7)

HRP B,C (pH 7)

HRPA-I (pH 10)

HRP B,C (pH 11.5)

HRP Compound II

HRP Compound X

1631 1 630 1622

1629 1627 1625

1640 1631 1620 1605

1 574 1573 1 550

1 566

1635 1630 1619 1603 1585 1 582 1 562 1 563

1635 1632 1620 1602

1 574 1 572 1549

1637 1630 1620 1605 1 586 1584 1 558 1564 1 505

1 507

1 507

1 510

1 475 1427 1401 1 401 1379 1340 1 305 1237

1 482 1428

1432 1400 1406 1378 1 341 1311 1 239

1 428

1491 [1505 ]

1499

t'3 Alg

P

P28 B2g 8~(=CH2) (1) v29 B2g U2o A2g

dp dp dp ap p ap ap dp

1429

1428

1399 1 374 1 340 1305 1 241

1399 1 374

dp

1 172

v4

Alg

8~(--CH 2) (2) v21 A 2~ P13 Big 1'(C b-C,0 (1) (U3o, u39) I'22 A2g

1 303 1237 1 169

1 128

dish peroxidase-cyanide With both sets of isoenzymes excited at 406.7 nm. High ~4 frequencies have been previously observed for compound II of horseradish peroxidase [23] and the myoglobinH 202 complex [24]. Discussion There are several interesting features in the resonance Raman spectra of horseradish peroxidase that are reported in this paper. In Fig. 1, under 406.7 nm excitation a strong polarized band appears in the range from 1627 to 1632 c m - 1 The prominent band with the highest frequency in protoporphyrin IX hemes is normally ul0 (B~g), which is depolarized. Through polarization measurements we found ul0 to be a weak feature to the high energy side of the strong polarized band which can be seen as a shoulder in many of the spectra shown in Fig. 1. The frequency of u~0 seen under 406.7 nm excitation is found to be in the range 1630 to 1640 cm -1, in agreement with the

1 171 1 137

1585

1378

1584 1 563

1 399 1 379

1 176 1 136

values seen in Fig. 2 under a-fl excitation, where Plo is the highest energy band and is strongly enhanced. (The differences in the labelled frequencies of Plo in Figs. 1 and 2 are a result of the difficulty in locating the U~o frequencies in Fig. 1 which appear as shoulders.) We assign the strong polarized band therefore to a u c _ c vinyl mode seen by Choi eta 1. [18,19] in infrared absorption spectra of nickel protoporphyrin. This assignment is not entirely certain, since, for the two Uc=c vinyl modes seen that have been reported [18,19], one is Raman active and the other is infrared active. We apparently see both modes in our Raman spectra (Table I and Fig. 1). An alternative possibility is that this band is a resonance enhanced histidine mode. Imidazole modes have been observed around 1630 cm -1 [25-27]. However we have not been able to observe any other modes assignable to a histidine imidazole. Thus, we favor the assignment of induced Raman activity into the infrared-active Uc c vinyl mode, to the strong polarized band seen between 1627 and 1632 cm-~, under Soret excita-

84 tion (406.7 nm). This is observed simultaneously with the Raman-active Pc:c vinyl mode at 1619-1625 cm -1. Isoenzyme A-1 at p H 7, exhibits a doublet at 1491 and 1505 cm -1 (both bands are polarized, Fig. la), which is not entirely evident for isoenzymes B and C that show a single band (Fig. lb) at 1499 cm -1. A smaller splitting of this band in isoenzyme C has been previously reported [28] under high resolution, and was found to be unaltered when the temperature was lowered. We suggest that the doublet is 1,3 (Alg) split by some sort of interaction (such as a Fermi resonance with a combination band). The reasons are as follows. First, no other A]g mode is expected in this particular frequency region [17-19]. Secondly, none of the other high energy bands is split, indicating the absence of the spin-state equilibrium that is observable at low temperature [29-34]. This is in contrast to the spin-state mixture seen in the resonance Raman spectra of alkaline met-hemoglobin [35] and alkaline met-myoglobin [36]. Finally, using the known correlations between porphyrin core size and the high-frequency skeletal modes [19], a core size of 0.201 nm can be calculated which, within the accuracy of the correlation parameters, would result in a single expected frequency of ~'3 at the center of the doublet at 1498 cm 1. We however make the suggestion of a Fermi resonance with extreme caution, since this particular splitting has not been previously observed in numerous model heme resonance Raman studies. The splitting may still be possibly due to a six-coordinate low-spin heme component of a spin state equilibrium, which cannot be ruled out. The other expected low-spin heme bands at 1564 and 1584 cm 1 may be hidden under the large 1572 cm -1 band, though if this were the case one would expect these additional low spin bands to be as readily apparent (as shoulders) as the strong 1491 and 1505 cm 1 bands. From the resonance Raman spectra shown in this paper, it is seen that the u4 frequencies of horseradish peroxidase Compounds II and X, which are believed to contain low-spin Fe(IV) hemes (Compound II is believed to contain an FelV=O [37], while Compound X has been proposed to contain an F e l V - o c 1 [13]), are not significantly higher than the u4 frequencies of the

alkaline forms of horseradish peroxidase isoenzymes which contain low-spin Fe(III) heroes. Other species believed to contain Fe(IV) hemes, such as the enzyme-substrate complex of cytochrome c peroxidase, and compound II of horse blood catalase, have been reported to have 1'4 frequencies of 1375 and 1376 c m - l , respectively [38]. These data suggest that high u4 frequencies may not be totally reliable indications of Fe(IV) oxidation states in hemes. While present experimental evidence does favor an empty sixth position in neutral peroxidase, the question remains as to the identity of the sixth ligand in the alkaline form. A water molecule or halide ion as the sixth ligand can be ruled out, since they are a weak field ligands, resulting in a high-spin rather than low-spin heme. Such examples are aquo-methemoglobin, aquo-metmyoglobin, horseradish peroxidase fluoride, metmyoglobin fluoride, and methemoglobin fluoride. These proteins exhibit resonance Raman frequencies for PlO' //19' and ull at 1610, 1583, and 1561 cm i [20] which are typical for six-coordinate high-spin hemes, and are much lower than the typical lowspin frequencies of the alkaline form of horseradish peroxidase shown in Figs. lc, ld and 2c. Ellis and Dunford [9], assuming the presence of a water molecule in the sixth position, have discussed ionization of the water to a hydroxide ion at alkaline pH, in analogy to the ionization of the water molecule to hydroxide observed in the iron(Ill) heme of aquo-metmyoglobin and aquo-methemoglobin [39]. Epstein and Shejter [40] and Iizuka et al. [41] have challenged this and have suggested a protein conformational change in the horseradish peroxidase transition based on kinetic measurements. Their measured rates were much lower than those measured for the alkaline ionization of aquo-methemoglobin and aquo-metmyoglobin, but similar to the alkaline ionization of ferric cytochrome c, which is believed to involve a protein conformational change [42]. Epstein and Schejter [40] and Iizuka et al. [41] have proposed that the peroxidase transition is not a proton dissociation of an iron-linked water molecule, but rather a deionization of a protein side-chain followed by a protein conformational change possibly allowing a protein side-chain to assume the sixth coordination position of the iron, resulting in a spin transi-

85 tion. T h i s has b e e n s u p p o r t e d b y r e s o n a n c e R a m a n studies w h i c h s h o w a s t r o n g F e - O H s t r e t c h at 490 c m -1 [43] in a l k a l i n e m e t m y o g l o b i n , w h i c h has b e e n r e p o r t e d to b e a b s e n t in a l k a l i n e h o r s e r a d i s h p e r o x i d a s e [28]. A m i n o acids s u c h as lysine, h i s t i d i n e , m e t h i o n ine or a r g i n i n e c o u l d , in p r i n c i p l e , t a k e the sixth p o s i t i o n . O f these, lysine is a c a n d i d a t e , as a P4 v a l u e of 1378 c m - 1 h a s b e e n r e p o r t e d for a l k a l i n e c y t o c h r o m e c [44] b e l i e v e d to c o n t a i n a lysine [45,46] at t h e sixth p o s i t i o n r e p l a c i n g a m e t h i o n i n e in the n e u t r a l e n z y m e , t h o u g h this h a s b e e n q u e s t i o n e d [47,48]. H o w e v e r , the r e p o r t e d 1378 c m -1 f r e q u e n c y for ~4 o f a l k a l i n e c y t o c h r o m e c was o b t a i n e d at 77 K [44]. W e h a v e o b s e r v e d , at r o o m t e m p e r a t u r e , a ~4 v a l u e o f 1375 c m - t for a l k a l i n e c y t o c h r o m e c ( S i g m a t y p e VI, 0.01 glycine, p H 11.0, 406.7 n m e x c i t a t i o n ) . Similarly, n e u t r a l c y t o c h r o m e c ( p H 7.0, 0.01 M p h o s p h a t e ) w i t h m e t h i o n i n e as the sixth ligand, was o b s e r v e d to h a v e a u4 v a l u e o f 1372 c m -1. 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 h a v e s u g g e s t e d t h a t n e i t h e r lysine [49] n o r h y d r o x i d e [50] m a y b e t h e sixth l i g a n d in a l k a l i n e h o r s e r a d i s h p e r o x i d a s e . A h i s t i d i n e as the sixth l i g a n d w o u l d n o t b e e x p e c t e d to result in a h i g h v4 f r e q u e n c y . T h e r e l e v a n t m o d e l c o m p o u n d [ I m 2 F e m P P ] C I ( I m = i m i d a z o l e ; PP = p r o t o p o r p h y r i n I X ) has a ~4 f r e q u e n c y o f 1373 c m -1 [19]. F u r t h e r studies will b e n e c e s s a r y to i d e n t i f y c o n c l u s i v e l y the sixth l i g a n d in the i s o e n z y m e s o f alkaline horseradish peroxidase.

Acknowledgements T h e a u t h o r s w o u l d like to t h a n k P r o f e s s o r s J o s e p h T o p i c h , P a u l Stein a n d T h o m a s G . S p i r o f o r s t i m u l a t i n g discussions, Mr. K e n t K o l l e r for a s a m p l e of p u r i f i e d c y t o c h r o m e c, a n d P r o f e s s o r F r e d M. H a w k r i d g e for r e a d i n g the m a n u s c r i p t . F i n a n c i a l s u p p o r t to J. T e r n e r is a c k n o w l e d g e d f r o m the R e s e a r c h C o r p o r a t i o n , the d o n o r s of the P e t r o l e u m R e s e a r c h F u n d as a d m i n i s t e r e d b y the A m e r i c a n C h e m i c a l Society, a n d the J e f f r e s s M e m o r i a l Trust.

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