1H-NMR characterization of cucumber peroxidases

3b

g&~'hwaica ~'t Bk~,~ftvsica Acta. 1118 ( 1991 ) 36-40 ,: It,~! El~.",i¢~ St'~ence Publi~be~ B.V. A]! r~ghl~r e . f r e d {}167-483/,I/91/c~O3~qfl

BBAPRO~ll4~

IH-NMR characterization of cucumber peroxidases L B . D u g a d ~. H . M . G o f f ~ a n d F.B. A b e l e s -" :

l)~'purtm~'nr . f Cttcmt~t~', Utz;.t~'~iO- i~f bm'a. bm'a ~'~.7,~. !.-1 ~ _ 4 . ~ apJ -~ U-~J_l~'lrdrlnlett! of A~nadmr~: .4gritullaral Re~earch .$e~twe..4ppalachtan Fmi~ Pa~'an-h StatUre. K~*arr~~ tll~; ||~" 11:.S..4.t (Retched 3 June I~1~

Key ~t~rd~: NMR. t tt-: Pen~xida-,<: Heine: Actr.-e si~e

Two peroxidase isoenz~'mes from Cucumber seedlings, one acidic ( p l - - 4 ) and one basic (pl--9), were characterized i~ tH-NMR spectroscopy. The NMR spectra were obtained in the native (ferric high.spin) and cyanide ligated (ferric low-spin) forms of both isoen~mes. The NMR spectral comparison of paramagnetically shifted resonances ~ith those of the well chm~,cterized horseradish peroxidase C, HRP(C), isnenzyme indicates that both cucumber peroxidases have a protohemin IX prosthetic gnmp ~ith proximal hislidine coordinated to the heine iron. The do'onfield heine IH-NMR shift pattern is distinct for each isoenz)me, and this reflects presumably dissimilar heine active site environments. The basic isoenz~me shows less asymmetry in heine gH-NMR signals as compared to the acidic isoenzyme or HRP(C} isoenzyme. It was also found that the acidic cucumber peroxidase exists predominantly as a monomeric species in solution with 30 kDa molecular mass as opposed to its earlier characterization as a 60 kDa dimeric protein.

Introduction Peroxidases are important en~mes responsible for the oxidation of a varie~' of organic and inorganic substrates among highly diverse organisms [I-3]. An understanding of the structure-function relationship of peroxidase en~xaes is of continuing interest. Previous work has shown that plant peroxidases play a role in ethylene-induced senescence of plant tissues [4.5]. The biosynthesis of these peroxidases is induced in plants by ethylene. Two ethylene-induced peroxidase isoenzsxaes, one acidic (60 kDa) and one basic (33 kDa). have been isolated from cucumber seedlings [4.5]. In this paper we report a preliminary. NMR characterization of these peroxidases in order to discern if ethylene-induced i s o e n ~ e s are structurally related to other plant peroxidases. The NMR spectral comparison with the well characterized HRP(C) isoenzyme made it possible to gain an insight into the active site structure of cucumber peroxidase isoenzy,rues that show

Abbreviations: CUPO. cucumber peroxidase: HRP(C). horseradish peroxidase C isoen~'me; NMR. nuclear magnetic re~nance: pl. ist~electric pH. Correspondence: ll.M. Goff. Dcparlment of Chcmisto-. University of Iowa, Iowa City. IA 52242. U,S.A.

fundamental spectral similari~" with the HRP(C) cn~Tae.

Materials and Methods

The peroxidase isocr~'mcs were isolated from cucumber seedlin~ as described earlier [4_S]. The proteins {2~S mg of the acidic and 5 mg of the basic isoen~'me) ~ere dissoh, ed in 0_'; ml ~9.8% D,O that contained 10 mM phosphate buffer (pD = 8), and the solution was transferred to a 5 mm NMR tube. The concentrations of acidic and basic isoenzymes were 0.15 and 0.3 mM, respectively, based on 30-33 kDa molecular masses. A sample of HRP(C) isoenzyme (from Sigma, T ~ e VI) was also prepared (1 mM conccntration) for comparative study. The low-spin ferric complexes were prepared by addition of a small (less than 0.5 rag) quantiD, of solid NaCN to the native pcroxidase samples. IH-NMR spectra were recorded on a Bruker AMX600 spectrometer operating at 600.14 MHz. The spectra of native isoenz~xnes were recorded with a 2K or 4K data size over a ~ 90 KHz spectral width with a 12 bit digitizer. The pulse repetition rate was 80-160 per s and 40K to 60K transients were collected. The free induction decays were zero filled to 16K data points and exponentially multiplied by 100 Hz line broadening

37 solution, confirming its assignment as a slowly exchanging proton. The spread of the heme methyl chemical shifts is less for basic CUPO as compared to both acidic CUPO and HRP(C) isoenzymes (Fig. 1). The heine methyl shift dispersion is associated with heme plane asymmetry in synthetic model heroes as well as hemoproteins [7]. Thus, the heine environment is different in the two CUPO isocnzymes, perhaps due to protein contacts a n d / o r slight orientation differences of the axial imidazole ligand. The heme methyl linewidths are smaller by 200-300 Hz for both CUPO isoenzymes as compared with those in the HRP(C). This is consistent with the molecular masses of the two CUPO is~nzymes in the 30-33 kDa range as compared to HRP(C) with a 42 kDa value. Hence, the linewidths for contact shifted resonances in high-spin ferric proteins have a contribution from a 'Curie-spin' relaxation mechanism that depends on the overall molecular correlation time at constant magnetic field and temperature [8]. The earlier characterization of CUPO indicated the molecular mass of 33 kDa for basic and 60 kDa for a dimeric acidic isoenzyme, with the possibility of dissociation of the acidic CUPO into monomers [4]. The NMR spectra clearly show the presence of predominantly monomefie acidic CUPO. Spectral information is available that is reIevant to the occupation of the sixth coordination site of the iron

prior to Fourier transformation to improve the signal to noise ratio. JH-NMR spectra for the low-spin complexes were acquired with 8K data points over a 40 KHz spectral width with a 16 bit digitizer. A standard solvent presaturation pulse sequence was used with a repetition rate of 1.6 per s. and 2-3K scans were collected. The spectra were obtained by 20 Hz exponential multiplication of free induction deca~. The spectra with a rapid repetition rate ( = 100 per s.) were obtained with a 50 KHz s-~eep-width from IK data points and 40K transients. The FIDs were zero filled to 8K and apodized with 40 Hz line broadening before Fourier transformation. Remits and Discussion The ~H-NMR spectra of natk'e CUPO isoenzymes are shown in Fig. IA and lB. The spectrum of HRP(C) isoenzyme, recorded under the same conditions, is shown in Fig. 1C for comparison. In all three spectra the most downfield shifted broad resonance of single proton intensity is obser~'ed in the 95-100 ppm range. This peak has been assi~ed in HRP(C) to the ring-NH exchangeable proton of the coordinated histidine residue [6]. The proton signal for CUPO isocnzymes showed a decrease in intensity after one week in D:O

-CH3

,I 7i CH3 -CH3 -CH3 "CH3 " . n

"----'-~ -CI13 -CH3

-cn3 II ,t

'

t

1@0

"

'

'

I

80

"

°

'

X1/8

-C!13 n

tl

I

60

~

1 ~

;

I

"

~

"

I

I

'

'

Xl/8 '

t

'

40 211 0 -20 PPM Fig. I. The (gl0MHz IH-NMRspectra of native peroxidasesin D,O. 298o K and pH = ,~.0.(A) acidicCUPO,(B) basic CUPO and (C) HRP(C) shmving hypedineshifted resonances.The heme methylpeaks in each spectrum are labelled. Nol¢ the presenceof a slowlyexchanging-NH proton fromproximalhislidinein the 95-100 ppm regionin each spectrum.

38 center of CUPO isoenz~.~es. A broad feature in the - 15 to -3~l p[m, upfield region spectra for the CUPO isoen~..'me is best ascribed to the heme meso protons. The meso proton signals have been observed in the upfield region for fr;e-coordinate heine model compounds [9.10]. and also in the deuterium NMR spectra of native HRI~C) [11]. This suggests that the basic isoenz~e, tike other peroxidases, is frye coordinated. The small quantity of available acidic isoen~me and resulting [o~'er sensit/vi~' precluded the observation of such a broad feature in the upfield region of this protein. The spectra of femc low-spin ~'anide complexes of CUPO isozymes are shown in Fig. 2A and B. The t H-NMR spectrum of the analogous HRP~C) complex is shown it~ Fig. 2C for comparison. The low-spin ferric complexes yield much better resolved hyperfine-shifted signak that have been correlated with active site structure of several perofidases [12-15]. There are two heine methyl peaks observed in the ~ - 3 5 ppm downfield region for each peroxidase engine (Fig. 2). It is noted that the chemical shift difference between the t~o methyl peaks of the basic CUPO is ve~, small as compared to an acidic CUPO or HRF(C) isoenzyme,

a~in reflecting differences in imidazole ring orientation or heine-protein contacts. In the upfield spectral re#on both CUPO Lsoenz~messhow a resolved peak of sin#c proton intensity in the - 7 to - 12 ppm region. The HRP(C) protein s h o ~ two peaks of one proton intensity in the -:5 to - 10 ppm region earlier assigned to an Arg amino acid residue [I3]. Limited quantities of protein preclude absolute assignment of CUPO peaks in this region, ahhou~ the single proton peak could weli be associated with a distal Arg residue. It is apparent that the two downfield heine methyl peaks in Fig 2A have unequal intensity. The relative intensity measuremenLs show that the methyl peak at 27 ppm has one additional peak of single proton intensity under the methyl peak. The presence of these additional hypeffine shifted peaks with a peculiar pauern suggests that two distinct forms of the protein are present. Given the precedem for dimerizadon of the acidic isoenz~me [4]. the presence of dimede protein would be log/cal. However. the species appear to be monomedc, based on the linewidth comparisons with the basic CUPO isoenz~ne and HRP~C). The spectra of low-spin cyanide complexes of CUPO recorded with a faster repetition rate are shown in Fig.

p r

"CH3

I

"CH3"Cit3

.CH 3

30

~

-CH3

:

j

20

I 10

! 0

'

V -10

PPM Fig. 2. The 60(I MHz ~H-NMR spectra of ferric low-spin cyanide complexes of pcroxidases in D : O at 298 ~ K. pH ~ 8.0. (A) acidic CUPO. (B) basic CUPO and (C) HRP{C}. The downfield heine methyl peaks are labelled in each spectrum.

39

-OH 3

,

q tt

C2-H

C2-H

.A._ 30

20

10

-10

-20

-30

PPM Fig 3, The 600 Mllz ~H-NMR spectra of ferric lo~v÷spino'anide complexesof perosidases acquired with a fast repetition rate ( = 100 per s) at 298 ° IL in D:O at pt! = 8, (At ac/dic CUPO. (BI b~sic CUPO and (C) HRP(CI i~zs'me. The fast relaxing histidine ring C4H and C:H proton pea~ ate labelled in each spectrum AnIh¢ dmvnfield and upfield spectral regions, respectively.

3A and B. These spectra were recorded to search for fast relaxing coordinated histidine ring C , H and C~H protons which show characteristic chemical shifts, Such proton peaks have been detected earlier for HRP(CL Coprinus macrorhizus peroxidase, lacto- and myeloperoxidase [12-15]. Broad proton signals are observed in the upfield - 2 5 to - 3 0 ppm region and in the downfield 20 to 25 ppm region for both C U P O isoenzymes (see Fig. 3), The downfield C a l l histidine proton is observed at - ~ ppm in all cases. In HRP(C) the signal is present under other peaks in that region (Fig. 3C), while both C U P O isocnzymes show a clear shoulder to the slow relaxing peaks. The C2H proton for both acidic C U P O and HRP(C) is observed at - 3 0 ppm, while for basic C U P O it is observed at - 2 7 ppm. These results strongly argue for the presence o f a coordinated proximal histidine in both C U P O isoenzymes. In conclusion, the spectral features of both C U P O isoenzymes show general similarities with those of HRP(C). This result is perhaps not surprising in view o f 53% sequence homology and conserved home region sequences bet~veen HRP(C) and a cucumber peroxidase e D N A clone [16]. The acidic and basic C U P O isoenzymes possess an axial histidine ligand and very likely a distal hisfidine residue. Spectral differences among the three proteins examined here reflect more

subtle changes in imidazole coordination and perturbations in the protein active site environment. Acknowledgement This work was supported by NIH grant G M 28831.

References I Dunford. H.B. and Stillman, LS. (]976) Coord. Chem. Rev. 19,

187-~1. 2 Dunford, H,B, (19821 Adv. Inorg, Biochem. 4. 41-68. 3 Van Huystee. R.B. (19871Annu. Roy. Plant Physiol.38. 205-219. 4 Ab~les. F.B., Dunn. LJ,, Morgans. P.. Callahan, A., Dinterman, R.E. and Schmidt, J. (19881 Planl. Physiol. 87. 609-615. 5 Ab¢les. F,B,, Hershberger, W.L. and Dunn. L.J. (1989} Plant. Physiol. 89. 664-668. 6 La Mar. G.N. and Dc Ropp, LS. (1979) Biochem. Biophys. Res. Commun. 90, 36-41. 7 La Mar. G.N. (1979) in Biological Applications of Magnetic Resonance (Shulman, R.G., ed.), pp. 305-343. Academic Press. New York. 8 Gueron. M. (1975) J. Magn. Reson, 19, 58-66. 9 Goff, H,M. and Shimomura, E. (19801 J. Am. Chem. Soc, 102. 31-37, !0 Budd, D.L., La Mar, G.N., l,angry, K.C., Smith. K.M. and Nayyir-Mazhir, R. (1979) J. Am, Chem. Soc. 101, 6091-6095. II La Mar. G.N., Thanabal. V., Johnson. R.D,, Smith. K.M. and Parish. D.W. (19891J. Biol. Chem. 264. 5428-5434.

40 1" LukaL G~S. Ro~gcts. K.R., Jalno. M.N. and Goff. H.M. (19~i9) B~ch¢raht~' ~'L 3338-3345. t3 Tha~baL V_. De P,t~p. J.S_ arm La Mar, G.N. ( ! ~ 7 ) J. Am. Chem_ So~-. ltD. 7516-75~. I4 ThanabaL V. arid La Mat. G.N. (1989) B/oct'.emist~" ~g. 7l)3S7044.

15 Du~e.~d. LB~ La Mar. G.N_ Le¢. H.C~ lkcda-Saito. M.. Booth, KS. and Cau~rh¢ . W.S. O9901J. B/oL Chem. 265. 7173-7179. 16 M~x,gcm. P H_ Calhhan. A.M.. Dann. L.J. and Abeles, F.B. (lq~g~ Plan,, MoL ~ . 14. 715-7"~-~.