HRP Depolarization Ratio in Organic Solvents: Enzyme Activity Analysis, Papers of Introduction to Sociology

Download HRP Depolarization Ratio in Organic Solvents: Enzyme Activity Analysis and more Papers Introduction to Sociology in PDF only on Docsity! Biophysical Journal Volume 84 May 2003 3285–3298 3285 Heme Structural Perturbation of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy Qing Huang,* Wasfi Al-Azzam,y Kai Griebenow,* and Reinhard Schweitzer-Stenner* Departments of *Chemistry and yBiology, University of Puerto Rico, Rı́o Piedras Campus, San Juan, Puerto Rico 00931-3346 USA ABSTRACT The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B1g distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme’s pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B2g-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP. INTRODUCTION Studying enzymes in nonaqueous solvents has emerged as a vivid and developing research area in biochemistry and biotechnology. This stems from the fact that many enzymes show new properties in organic solvents, i.e., the capability to catalyze reactions that do not take place in water (Klibanov, 1997) and solvent control of the stereoselectivity (Klibanov, 1990; Faber et al., 1993; Schoffers et al., 1996). However, enzymes are typically far less active in non- aqueous solvents than in the aqueous environment (Kliba- nov, 1997). This has resulted in enzyme activation in organic solvents being attempted by many groups using various strategies (see Griebenow et al., 1999, and references therein). One main difficulty in assessing the reasons for the changed enzyme activity in organic versus aqueous environment is that many factors influence enzyme activity in organic solvents, e.g., enzyme structure, conformational mobility, substrate and product solubility, and transition state solvation (Schmitke et al., 1996; Klibanov, 1997; Griebenow et al., 2001). In most applications, enzymes are used as dispersed dehydrated powders in organic solvents and more than crude secondary structure data have not yet be- come available to be related to the activity (Griebenow and Klibanov, 1997). Various methods used to solubilize en- zymes in organic solvents (Paradkar and Dordick, 1994; Mabrouk, 1997) have become relevant in this context because they allow an assessment of the relationship between secondary and tertiary structure and enzymatic activity in these solvents (cf. e.g., Secundo et al., 1999). Generally, the fine structure of the respective active site cannot be explored under these conditions. Horseradish peroxidase (HRP), however, is an exception from this rule, because its active site is constituted by a chromogenic prosthetic heme group that can be selectively probed by resonance Raman spectroscopy. HRP has been widely employed as model enzyme in nonaqueous enzymology (see e.g., Kazandjian et al., 1986; Dordick et al., 1986; Gorman and Dordick, 1992; Ryu and Dordick, 1992) and it is involved in a variety of chemical reactions (Adam et al., 1999). Upon modification with poly(ethylene glycol) (HRP- PEG) and subsequent dissolution in benzene and toluene, the enzyme adopts a structure very similar to that in aque- ous solution (Mabrouk, 1995; Mabrouk and Spiro, 1998; Al-Azzam et al., 2002). HRP is a model system for exploring the enzymatic mechanisms of class III peroxidases and the underlying structure-function relationships due to its availability, re- markable catalytic activity, and stability in its natural aqueous environment as well as in a variety of organic solvents (Dunford, 1999; Kazandjian et al., 1986; Gorman and Dordick, 1992). The crystallographic structure of the wild-type of the most abundant isoenzyme HRPc and its complex with the model substrate benzohydroxamic acid (BHA) is well known (Fig. 1) (Gajhede et al., 1997; Henriksen et al., 1998). Covalent HRP modification with poly(ethylene glycol) greatly increases the solubility and reactivity in both hydrophilic and hydrophobic organic solvents, yielding homogeneous, optically transparent solu- tions (Takahashi et al., 1984; Mabrouk, 1995; 1997). HRP- PEG and its enzymatic intermediates in organic solvents have much better photostability and thermostability than in Submitted September 6, 2002, and accepted for publication January 3, 2003. Address reprint requests to Reinhard Schweitzer-Stenner, Dept. of Chemistry, University of Puerto Rico, Rı́o Piedras Campus, PO Box 23346, San Juan, PR 00931-3346 USA. Tel.: 787-764-2417; Fax: 787-756- 8242; E-mail: rstenner_upr_chemistry@gmx.net.
2003 by the Biophysical Society 0006-3495/03/05/3285/14 $2.00 aqueous solution (Mabrouk, 1995) so that they become amenable to spectroscopic studies. It has been shown that enzymatic activity and structural integrity of lyophilized HRP is retained in various organic solvents, especially in benzene and toluene (Kazandjian et al., 1986). The structure of dehydrated HRP-PEG dis- solved in organic solvents essentially resembles the structure of native HRP in water (Ryu and Dordick, 1992; Kamiya et al., 2000; Al-Azzam et al., 2002). Despite the striking similarities in structure and enzymatic activity, however, special properties have been reported for HRP in some aromatic organic media, such as BHA (Teraoka and Kitagawa, 1981) and benzene (Mabrouk and Spiro, 1998). Akasaka et al. (1995) showed that HRP efficiently catalyzes the hydroxylation of benzene in the presence of H2O2, similar to what was obtained for cytochrome P450. Since the redox potential of benzene is higher than that of HRP in all its intermediates, an electron transfer can be ruled out as causing this reaction. Akasaka et al. found that 95% of the oxygen incorporated into the phenol product was from peroxide, a finding consistent with an oxo transfer mecha- nism. From their resonance Raman data, Mabrouk and Spiro (1998) proposed that such a mechanism requires the benzene molecule to slip into the heme pocket where it has to be located adjacent to the ferryl oxygen of compound I and thus also in close proximity to the heme group. They further hypothesized that this process is facilitated by the following structural alterations in the heme pocket: 1), A benzene molecule is bound at Phe179, which perturbs Gln176 and disrupts its H-bond to the heme propionate substituent. 2), A salt bridge is formed between one propionate and distal Arg38. 3), The imidazole residue of His170 moves toward Asp247 more closely, thus weakening the Fe-Ne(His 170) bond. If this view of the HRP-benzene complex is correct, the interacting benzene molecule shall significantly perturb the symmetry of the heme macrocycle. Such a perturbation has already been identified for HRP-PEG:BHA complexes, which in the hexacoordinate ferrous state exhibit significant Q-band splitting at cryogenic temperatures (Kaposi et al., 2001). Since benzene is expected to be closer to the heme group, its symmetry-lowering deformation efficiency should be even stronger than that of BHA. Such deformations can be obtained by means of polarized resonance Raman dis- persion spectroscopy (PRRDS) (Schweitzer-Stenner, 1989; Schweitzer-Stenner, 2001). This technique determines static normal coordinate deformations (SNCDs) introduced first by Schweitzer-Stenner et al. (1984) and later in greater detail by Shelnutt, Jentzen, and associates (Jentzen et al., 1998; Shelnutt, 2000). The concept roots from the fact that any distortion in the porphyrin macrocycle can be approximately described as the superposition of several basic deformations along the normal modes of the unperturbed macrocycle in its D4h symmetry. For planar distortions of the heme macro- cycle, the deformation types can be attributed to the sym- metry representations A1g, B1g,A2g, and B2g of theD4h point group (Shelnutt, 2000; Schweitzer-Stenner, 1989). PRRDS is an excellent probe of SNCDs. The technique involves the determination of the depolarization ratio dispersion (DPR) of intense and structure sensitive marker bands of the Raman spectrum. Whereas the DPR is independent of the excitation wavelength for the ideal D4h symmetry, the SNCDs give rise to a mixing of different symmetries into the Raman tensor, which combined with interferences between different vibronic coupling mechanism causes the DPR to depend of the excitation wavelength (Schweitzer-Stenner, 2001). In this study, we applied PRRDS to HRP-PEG dissolved in benzene and toluene and HRP-PEG:BHA in water. Polarized Raman spectra were measured with different excitation wavelengths covering the Qv-band region of the optical spectrum. The data were analyzed by invoking basic group theoretical arguments to obtain changes of specific asymmetric heme distortions. We show that aromatic solvent molecules and BHA induce distortion of different symmetry types due to their different locations in the heme pocket. Particularly our data provide conclusive evidence that a benzene and a toluene molecule are located close to the heme group, in full support of the model suggested by Mabrouk and Spiro (1998). Our results led us to hypothesize that the symmetry-lowering distortion of the heme might decrease the dissociation barrier for the ferryl ligand in the compound I state to facilitate the hydroxylation of benzene. FIGURE 1 Crystallographic structure of HRP heme environment (Gaj- hede et al., 1997, Henriksen et al., 1998). Downloaded from the Protein Data Bank (1ATJ). 3286 Huang et al. Biophysical Journal 84(5) 3285–3298 obtained in our previous investigation, for which a mixture of various HRP isoenzymes was employed (Al-Azzam et al., 2002). The spectral shifts are likely to result from electronic interactions between the respective solvent molecule and the Eg HOMOs of the macrocycle, which lower the energies of the latter and thus decrease the respective transition energy from the A2u and A1u LUMOs. Fig. 3 depicts a set of Raman spectra recorded between 1300 and 1700 cm
1 for ferric HRP and HRP-PEG in dif- ferent solvents with 457.9 nm excitation wavelength. The Raman spectrum of native HRP in aqueous solution is plotted in the bottom panel as a reference in which all characteristic heme bands, namely, n21, n4, n3, n11, n2, n19, and n10 can be clearly identified, in accordance with the assignment by Smulevich and co-workers (Smulevich et al., 1994; Howes et al., 2001). The band profiles in Fig. 3 result from the spectral decomposition described in Materials and Methods. The spectral parameters of the most relevant bands are listed in Table 2. A significant overlap of n10 with the bands of the C55C stretching modes of the two vinyl substituents at 1620 cm
1 and 1630 cm
1 (Smulevich et al., 1994) can be ruled out, because all three bands observed between 1615 and 1640 cm
1 are clearly depolarized (vide infra), whereas the localized and uncoupled vinyl modes (DeVito et al., 1992) give rise to polarized Raman bands. Unfortunately, an exact spectral analysis of the region between 1550 and 1600 cm
1 was rendered impossible due to the overlap of at least five different Raman bands. We heuristically decomposed this part of the spectrum into two representative bands at 1572 and 1583 cm
1 (Table 3). The effective wavenumbers of these two depend on the excitation wavelength since they result from overlapping contributions of n2/n19a and n2/n19b, respectively, where n19a and n19b represent conformers with different spin states of the heme iron (vide infra). All spectra were normalized onto the n4 band of HRP to compare the relative intensities of different Raman bands. HRP (Fig. 3, panel A) and HRP-PEG (panel B) in aqueous solution show no significant spectral differ- ences between their respective wave numbers and intensities. This corroborates the notion that the tertiary structure of the heme cavity is maintained upon poly (ethylene glycol) modification (Mabrouk and Spiro, 1998; Al-Azzam et al., 2002). However, when HRP-PEG was dissolved in benzene (panel C) and toluene (panel D), some spectral discrepancies became apparent. The bandwidth of n4 is broadened by ;3 cm
1. The relative intensities of n3 and n11 are reduced in benzene and toluene. Since this broadening does not change the Lorentzian character of the band, it most likely results from a smaller dephasing time due to increased coupling with low frequency modes of the environment (Asher and Murtaugh, 1983; Schweitzer-Stenner et al., 1993). The band profiles of n10 (Table 4) are similar in benzene and toluene, but slightly different from that of native HRP in aqueous solution. On the other hand, differences between the Raman TABLE 1 Spectral parameters obtained from a five-band fit to the absorption spectra of HRPc and HRPc-PEG in water (with and without BHA) and organic solvents HRP in water HRP-PEG in water HRP-PEG in benzene HRP-PEG in toluene HRP-PEG with BHA in water Peak [nm] Width [nm] A* Peak [nm] Width [nm] A* Peak [nm] Width [nm] A* Peak [nm] Width [nm] A* Peak [nm] Width [nm] A* 376 6 1 39 6 2 1417 376 6 1 41 6 2 1583 386 6 2 43 6 2 1025 384 6 2 44 6 2 1213 385 6 1 41 6 1 922 404 6 1 29 6 1 1723 404 6 1 30 6 1 1788 412 6 1 29 6 1 1377 412 6 1 30 6 1 1574 408 6 1 21 6 1 1361 504 6 2 49 6 1 153 503 6 1 49 6 1 175 523 6 1 47 6 3 69 523 6 1 37 6 3 61 507 6 1 45 6 1 108 537 6 2 27 6 2 27 536 6 1 30 6 2 35 546 6 2 43 6 3 46 548 6 2 35 6 3 54 542 6 1 26 6 1 15 645 6 2 48 6 3 138 647 6 2 41 6 3 122 650 6 2 47 6 2 84 650 6 2 45 6 2 77 639 6 1 45 6 1 130 *Integrated molar absorptivity (mM cm
1 nm). FIGURE 2 (A) UV-vis absorption spectra of HRPc ( full line) and HRPc- PEG (broken line) in 10 mM phosphate buffer at pH 7. (B) UV-vis spectra of HRPc-PEG with 5 mM BHA in Tris buffer at pH 8 ( full line), HRPc-PEG lyophilized from water pH 7 and redissolved in toluene (short dashes), and in benzene (dotted line). The insets show the region of Q absorption bands. HRPc in Organic Solvents 3289 Biophysical Journal 84(5) 3285–3298 spectra of HRP-PEG in benzene and in toluene are negligible. Remarkable differences are observed, however, for HRP-PEG:BHA for which n3, n11, n2, and n10 are all downshifted (panel E). Spin and coordination state To compare the iron spin and coordination states of the investigated samples, we first utilized the n3 band at around 1500 cm
1 because this band is well isolated and particularly spin sensitive (Spiro, 1985). In the spectra of native HRP, n3 contains at least two components at 1492 and 1500 cm
1, which represent coexisting protein conformations with their iron atoms exhibiting pentacoordinate high spin (pc-hs) and pentacoordinate quantum mixed spin (pc-qms) states, re- spectively (Feis et al., 1998; Howes et al., 2000, 2001). The weaker band at 1492 cm
1 was previously assigned to a hexacoordinate high spin (hc-hs) (Rakshit and Spiro, 1974; Teraoka and Kitagawa, 1981; Evangelista-Kirkup et al., 1985; Palanappian and Terner, 1989.; Smulevich et al., 1991) and has recently been reassigned as pentacoordinated high spin state (Feis et al., 1998, Howes et al., 2000). We also assign it to pc-hs because this is consistent with the observation of a n10 band at 1629 cm
1, which is diagnostic of a pc-hs state for HRP (Smulevich et al., 1991, Feis et al., 1998, Howes et al., 2001) and also for barley (Howes et al., 1999) and soybean seed coat peroxidases (Nissum et al., 1998). These conformers are very likely to exhibit different distances between the proximal imidazole and the heme iron and, thus, different strengths of the axial ligand field. The 1500 cm
1 line of the pc-qms state is more intense than the 1492 cm
1 line of the pc-hs state in native HRP. Apparently, this is still the case for HRP-PEG in benzene even though the band profile appears somewhat more asymmetric toward the low wavenumber side indicating a somewhat higher occupation of the conformation with a pc-hs state. In toluene, the judgment of n3 signal is more difficult because this band is masked by a signal at 1497 cm
1, which arises from neat toluene. Our results are somewhat at variance with the spectra of Mabrouk and Spiro, who found n3 to be downshifted from 1495 cm
1 to 1480 cm
1 in the spectrum of HRP-PEG in benzene. This would be indicative of a dominant hc-hs state. However, this discrepancy can be resolved by an analysis of the other spin marker lines. Although the existence of a conformer with a pc-qms state in HRP-PEG in benzene and toluene is verified by the observation of a n11 band at 1547 cm
1, its significantly reduced intensity is indicative of a much smaller fraction of the pc-qms conformer in benzene and toluene (Table 1). This notion is further corroborated by the intensity distribution of the three n10 bands at 1621, 1629, and 1637 cm
1 in the spectra depicted in Fig. 3, which are assignable to con- formers with hc-hs, pc-hs, and pc-qms states of the heme iron, respectively. For native HRPc, the fractional intensities with respect to the total intensity of all n10 bands are 0.18 (hc-hs), 0.49 (pc-hs), and 0.33 (pc-qms) (Table 4), whereas the corresponding values for HRPc-PEG in benzene are 0.34, 0.38, and 0.28. For HRPc-PEG in toluene, similar values FIGURE 3 Raman spectra of ferric HRP under different experimental conditions with 457.9 nm excitation. (A) HRP in water, pH 8; (B) HRP-PEG in water, pH 8; (C ) HRP-PEG in benzene; (D) HRP-PEG in toluene; (E) HRP- PEG:BHA in water, pH 8. 3290 Huang et al. Biophysical Journal 84(5) 3285–3298 were obtained (0.37, 0.38, and 0.25). This clearly shows that indeed the hc-hs conformer is somewhat stabilized in the employed aromatic solvents, although it still coexists with the two other spin and coordination states. (A direct deter- mination of the molar fraction of the three conformers from the fractional intensities of the marker bands is not possible, because it is likely that the respective resonance excitation profiles are different in the preresonance and resonance region of the Qv-band covered by the employed excitation wavelengths.) The difference between our n3 band profile and that reported by Mabrouk and Spiro (1998) can best be explained by the different excitation wavelengths employed. The 406-nm excitation used in their study can be assumed to predominantly enhance the hc-hs species because its ab- sorption maximum is very likely close to this wavelength. This is in agreement with the Soret absorption maximum at 408 nm of HRPc-PEG in the presence of BHA (Fig. 2 B), where a hexacoordinated species is predominant (see below). The absorption maximum of the pc-qms state can be ex- pected to be at a more blue-shifted position (Feis et al., 1998). Different from HRP-PEG in neat organic solvents, HRP- PEG:BHA in aqueous solution seems to predominantly exist in a single conformation. Single n3 and n10 bands were observed at 1491 cm
1 and 1618 cm
1, respectively. The bands at 1622 and 1630 cm
1 are now attributed to vinyl vibration owing to its depolarization ratio. The marker bands n2 and n11 are downshifted to 1568 and 1543 cm
1, TABLE 2 Spectral parameters of prominent marker bands in the resonance Raman spectra of native HRPc and HRPc-PEG in water and organic solvents taken with 457 nm excitation Wave number [cm
1] Bandwidth [cm
1] Fractional intensity Assignment Wave number [cm
1] Bandwidth [cm
1] Fractional intensity Assignment HRP in water HRP-PEG in water n4 1373 10.7 0.33 1373 10.7 0.32 n3 1493 11.2 0.03 pc-hs* 1493 11.2 0.02 pc-hs* 1500 13.0 0.10 pc-qmsy 1500 13.0 0.10 pc-qmsy n11 1547 12.5 0.08 pc-qms y 1547 12.5 0.07 pc-qmsy n10 1622 8.5 0.05 hc-hs y 1622 8.5 0.04 hc-hsy 1629 7.7 0.10 pc-hsy 1629 7.7 0.11 pc-hsy 1637 10.2 0.09 pc-qmsy 1637 10.2 0.11 pc-qmsy HRP-PEG in benzene HRP-PEG in toluene n4 1373 13 0.33 1373 15.6 0.45 # n3 1492 11.2 0.01 pc-hs* 1493 11.2 0.005 pc-hs* 1500 13.0 0.04 pc-qmsy 1500 13.0 0.03 pc-qmsy n11 1547 13.4 0.06 pc-qms y 1547 10 0.04 pc-qmsy n10 1622 8.5 0.09 hc-hs y 1622 8.5 0.09 hc-hsy 1629 7.7 0.08 pc-hsy 1629 7.7 0.10 pc-hsy 1637 10.2 0.08 pc-qmsy 1637 10.2 0.8 pc-qmsy The integrated intensities of the Raman lines are given as fraction of the total Raman scattering detected in the spectral region shown in Fig. 3. Spectral parameters of prominent marker bands in the resonance Raman spectra of native HRPc and HRPc-PEG:BHA in water HRP HRP-PEG with BHA n4 1373 13 0.33 1373 10 0.37 n3 1492 11.2 0.01 pc-hs* 1490 12.3 0.13 hc-qms z 1500 13.0 0.04 pc-qmsy n11 1547 13.4 0.06 pc-qms y 1543 7.8 0.04 hc-qmsz n10 1622 8.5 0.09 hc-hs y 1617 8 0.08 hc-qmsz 1629 7.7 0.08 pc-hsy 1622 8.2 0.05 vinyl§ 1637 10.2 0.08 pc-qmsy 1630 7.3 0.05 vinyl§ *Interference from a toluene band at 1379 cm
1. yHowes et al. (2001). zIndiani et al. (2000). §Smulevich et al. (1994). TABLE 3 Wave numbers (in units of cm21) obtained from the heuristic two-band fit to the region between 1550 and 1600 cm21 and assignments to overlapping bands from n2, n19, and n37 HRPc in water HRP-PEG in water HRP-PEG in benzene HRP-PEG in benzene HRP-PEG with BHA n2 1 n19 1 n37 Peak 1 1572 (n2 1 n19a) 1572 (n2 1 n19a) 1572 (n2 1 n19a) 1572 (n2 1 n19a) 1567 (n2) Peak 2 1583 (n19b 1 n37) 1583 (n19b 1 n37) 1587 (n19b 1 n37) – 1579 (n19) HRPc in Organic Solvents 3291 Biophysical Journal 84(5) 3285–3298 FIGURE 5 Simulation of DPRs for (a) A1glike, (b) A2glike, and (c and d) B1glike modes of hemes subject to rhombic distortions. Increase of rhombic distortion is represented by the cases (1), (2), (3) in sequence. For B1glike mode, changes of DPRs upon increasing solely B1g or B2g distortion are illustrated in the cases c and d, separately. 3294 Huang et al. Biophysical Journal 84(5) 3285–3298 and n21, however, might also reflect changes caused by the binding of a sixth ligand (i.e., H2O). Particularly the DPR of n4 is known to be very sensitive to interactions between the heme core and axial ligands (Schweitzer-Stenner, 1989). With respect to the sixth ligand, el Naggar et al. (1985) reported a significant and systematic increase of the n4 DPR upon oxygen binding to myoglobin. For HRP-PEG:BHA in water, however, the DPRs of n11 strongly suggest that the interaction between the substrate and the heme gives predominantly rise to B2g distortions. Overall, distortions are between those of HRP-PEG in water and in the investigated aromatic solvents, as expected. Our results are at variance with the deformations obtainable from a normal-coordinate structural decomposition (NSD) analy- sis of the wild-type HRP:BHA crystal structure, which in- dicate significantly reduced rhombic B1g and B2g distortions of the heme. On the other hand, however, our data are con- sistent with spectroscopic studies showing a Q-band splitting of ferrous low spin HRP:BHA-CO spectra, which is absent in the spectra of the corresponding HRP-CO species (Kaposi et al., 2001) and with electron paramagnetic resonance data that indicate that significant rhombicity of the ligand field is maintained upon BHA binding (Indiani et al., 2000). Three explanations can be given for this discrepancy. First, one can interpret them as indicating that the heme structure of HRP in aqueous solution is more distorted than in the crystal. This does not seem unlikely in view of the very flexible and open heme pocket. This notion is not at odds with the finding of Smulevich et al. (1999), who reported nearly identical Raman spectra for HRP:BHA in solution and in a single crystal, since DPR changes reflect variations of the first derivative of the potential surface along the displacement coordinates of low-frequency B1g and B2g modes, whereas frequency changes of a Raman active mode are due to changes of the second derivative of the potential surface with respect to its own normal coordinate. Second, it is in prin- ciple possible that the differences between the DPR values of HRPc-PEG and HRPc-PEG:BHA reflect structural differ- ences between the excited electronic states of the respective heme groups rather than between their ground states (Schweitzer-Stenner et al., 2000). Third, one has to take into consideration that the x-ray structure was obtained for genetically engineered HRP without any glycans (Henriksen et al., 1998), whereas all the spectroscopic work was done with HRP in its natural glycolysated form. Since resonance Raman spectra of Teraoka and Kitagawa (1981) reveal no changes of the 379 cm
1 Raman line of the propionate substituent, any interaction of BHA with the respective pyrrole groups are likely to be weak. The crystal structure (Henriksen et al., 1998) exhibits BHAnearly parallel oriented to the heme group and in van der Waals contact with a Cm atom of one of the methine bridges between the two pyrrole groupswithmethyl and vinyl substituents. This can be expected to give rise to an electronic perturbation of the macrocycle due to pp-interaction. As experimentally and theoretically shown for meso-substituted nitroporphyrins (Schweitzer-Stenner et al., 2001), such a distortion gives rise to B2g and an Eu distortions, in full accordance with our data. This interpretation is further corroborated by the fact that the reaction with BHA has a comparatively strong impact on the DPR of n21, which is mostly a CmH bending mode (Li et al., 1990). In a recent study on native HRPc (Huang et al., 2003), we have invoked group theoretical arguments to show that FIGURE 6 A schematic sketch of rhombic (a) B1g and (b) B2g deformations of the heme macrocycle. HRPc in Organic Solvents 3295 Biophysical Journal 84(5) 3285–3298 particularly a perturbation leading to B2g distortions of the heme can stabilize the intermediate spin state of the iron atom and concomitantly facilitate its interaction with the high spin state by spin orbit coupling (Maltempo, 1974). Maltempo and co-worker (1979) have argued that it should be easier to oxidize the qms then the hs state, which is important for the formation of compound I. Apparently, the protein conformer with a qms iron is still predominant for HRPc-PEG in the investigated organic solvents. For HRP- PEG:BHA, the strong increase of the B2g distortion might be the reason for the very rare hc-qms state inferred from electron paramagnetic resonance data (Indiani et al., 2000). If Maltempo et al. (1979) are right, one expects that the enzyme should exhibit significant enzymatic activity in benzene and toluene. This is in line with findings by Kazadjian et al. (1986). The functional role of the B1g distortion particularly obtained for HRPc-PEG in benzene and toluene is less clear, but it is likely that the respective perturbation also stabilizes the pc-qms state, though some- what more indirectly than B2g perturbations. SUMMARY Taken together, our data provide evidence that the structural integrity of the heme group is maintained when HRP-PEG is dissolved in organic solvents. Some structural changes, however, are induced by benzene and toluene molecules penetrating the heme pocket. This slightly stabilizes the hc- hs state of the heme iron with respect to the dominant pc-qms state observed for native HRP and HRP-PEG in the resting state. In agreement with the hypothesis of Mabrouk and Spiro (1998), our data strongly corroborate the notion that benzene as well as toluene are in close contact with one of the pyrrole groups (most likely that directed toward Phe179 and Gln176), which gives rise to an additional B1g type distortion. HRP-BHA in water shows a different mode of interaction in that the contact between BHA and a heme methin carbon gives rise to a B2g-type distortion and to a predominantly hc-hs state. Both distortions are likely to stabilize the qms state of the heme iron, which has been hypothesized to facilitate the metal’s oxidation in the compound I formation process. Overall, our studies show that substrate binding (also the aromatic solvent molecules function as substrates) causes changes of the heme defor- mation, and, of course, it is the thus induced structural state and not that of the native enzyme that determines the heme contribution to the overall enzymatic reactivity. 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