©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Resonance Raman Investigation of Cyanide Ligated Beef Liver and Aspergillus niger Catalases (*)

Jamil Al-Mustafa , Milan Sykora , James R. Kincaid

From the (1) Chemistry Department, Marquette University, Milwaukee, Wisconsin 53233

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Resonance Raman spectroscopy has been used to investigate the properties of cyanide-bound beef liver catalase (BLC) and Aspergillus niger catalase (ANC) in the pH range 4.9-11.5. Evidence has been obtained for the binding of cyanide to both BLC and ANC in two binding geometries. The first conformer, exhibiting the [Fe-CN] stretching mode at a higher frequency than the [Fe-C-N] bending mode, exists as an essentially linear Fe-C-N linkage. For both BLC-CN and ANC-CN, the [Fe-CN] and [Fe-C-N] frequencies of this conformer were practically identical and observed at 434 and 413 cm, respectively. The second conformer exhibits a [Fe-CN] mode at lower frequency than the [Fe-C-N] mode, and is thus characteristic of a bent Fe-C-N linkage. The [Fe-CN] and [Fe-C-N] modes were identified at 349 and 445 cm, respectively, for BLC-CN, and at 350 and 456 cm, respectively, for ANC-CN. The two conformers persist in the pH range 4.9-11.5. Furthermore, upon raising the pH to 11.5, the [Fe-CN] mode of the linear conformer of BLC-CN downshifts to 429 cm while that of the bent conformer remains unchanged. The observed pH-dependent shift is attributed to the deprotonation of a distal-side amino acid residue, probably a distal histidine. The Fe-C-N axial vibrations of the two conformers identified for ANC-CN did not show any significant pH-dependent shifts, indicating a more stable hydrogen bonding interaction relative to BLC-CN.


INTRODUCTION

Catalase (EC 1.11.1.6; HO: HO-oxidoreductase) is a heme protein found in nearly all aerobically respiring organisms. Although catalases can catalyze the peroxidative oxidation of a wide range of organic and inorganic substrates, its primary function is believed to be to protect the cell components from the hazardous effects of hydrogen peroxide by catalyzing its dismutation according to the reaction: 2 HO 2 HO + O. Catalases have been isolated from a wide range of organisms, including mammals (Sumner and Dounce, 1937), plants (Galston, 1957), fungi (Scott and Hammer, 1960), and bacteria (Herbert, 1948). Most isolated catalases are tetramers of noninteracting subunits with one heme group per subunit. The crystal structure has been solved and refined for three catalases: bovine liver catalase (BLC)() (Murthy et al., 1981), Penicillium vitale catalase (PVC) (Vainshtein et al., 1986), and Micrococcus lysodeikticus catalase (MLC) (Murshudov et al., 1992) at 2.5-, 2.0-, and 1.5-Å resolution, respectively. The crystal structure of the three catalases are very similar, the heme groups of each subunit being deeply buried in a nonpolar cavity formed by single polypeptide chains. The most distinctive feature of the catalase structure is the coordination of a phenolate group of the proximal tyrosine residue to the heme iron as a fifth ligand. Interestingly, attempts to model catalase activity with simple iron porphyrin compounds indicate that axial ligands with an oxygen donor ( e.g. phenolate, tyrosinate, alcoholate ligands) are less efficient in catalyzing the dismutation of hydrogen peroxide than ligands with nitrogen donors ( e.g. imidazole) (Belal et al., 1989; Robert et al., 1991). However, the coordination of these nitrogen donor ligands to the heme enhances not only the catalase activity but also the oxygenase activity, which is destructive for the distal amino acid residues of the protein (Belal et al., 1989; Robert et al., 1991). This oxygenase activity is quite small for oxygen donors, but they still maintain reasonable catalase activity.

The catalytic cycle of catalase involves the reaction with two molecules of hydrogen peroxide to form water and oxygen. The first step is an oxidation process involving the reaction with one molecule of hydrogen peroxide to form an oxidized reactive intermediate, known as compound I, and water. The second step is a reduction process which involves the reaction of compound I with a second molecule of hydrogen peroxide to regenerate the ferric catalase and molecular oxygen. Therefore, the mechanism of catalase action can be written (Schonbaum and Chance, 1976) as the following.

On-line formulae not verified for accuracy

Compound I of catalases, like that of HRP, is best described as a ferryl -cation radical, which is produced by the heterolytic cleavage of the O-O bond of hydrogen peroxide and it is well established that the active sites of peroxidases and catalases contain structural elements which facilitate the transition state charge separation and promote the heterolytic O-O bond cleavage (Fita and Rossman, 1985a; Finzel et al., 1984; Poulos, 1987). Recently, it has been reported that BLC binds four molecules of NADPH very tightly (Kirkman and Gaitani, 1984; Fita and Rossman, 1985b). Although the removal of the bound NADPH molecules does not eliminate the catalase activity, a significant loss of activity was observed upon lengthy exposure to HO. It has been proposed that binding of NADPH circumvents the inactivation of catalase by its natural substrate, hydrogen peroxide, by preventing the accumulation of compound II (Kirkman et al., 1987).

Resonance Raman (RR) spectroscopy is a powerful technique for the investigation of hemeprotein activesites (Spiro and Li, 1988) and axial-ligand vibrations (Yu and Kerr, 1984; Yu et al., 1988). Previous RR studies have concentrated on the investigation of the skeletal modes of the native forms of various catalases, the enhancement of the axial tyrosinate modes through excitation in the visible region (Chuang et al., 1988; Sharma et al., 1989; Nagai et al., 1989), and the detection of the reactive intermediates in the catalytic cycle (Chuang et al., 1989; Chuang and Van Wart, 1992). The lack of RR studies on catalases ligated with various exogenous ligands prompted us to carry out an investigation of the ligated forms of catalases in order to probe the heme distal environment and the effect of the endogenous tyrosinate ligation on the vibrational modes of the axial ligand. Recently, this group reported the results of an RR investigation of CO-ligated BLC (Hu and Kincaid, 1992). In other reports, we have demonstrated that RR investigation (Al-Mustafa and Kincaid, 1993; Hu and Kincaid, 1992) of the cyanide adducts of peroxidases can provide vital information about the important structural elements of the active sites and reveal the role of distal- and proximal-side heme environments on the catalytic cycle. In the present work, we report RR studies of the cyanide adducts of two catalases, a mammalian catalase (BLC) and a fungal catalase (ANC).


EXPERIMENTAL PROCEDURES

Materials and Methods

BLC powder was purchased from Sigma and purified by standard methods (Browett and Stillman, 1979; Eglinton et al., 1983; Kikuchi-Torii et al., 1982). BLC was dissolved in deionized water and centrifuged. The deep green solution was dialyzed for 6 h against deionized water and for 12 h against 20 mM potassium phosphate buffer (pH 7). The BLC solution was then loaded onto a DE-52 (Whatman) column (2.5 25 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7). The column was washed with 100-200 ml of the same buffer and eluted with a linear gradient consisting of 300 ml of 20 mM and 300 ml of 300 mM potassium phosphate buffer (pH 7). The BLC fractions having an Rz ( A/ A) value greater than 0.9 were pooled and used for spectral measurements.

Aspergillus niger catalase (ANC) suspension in 3.2 M ammonium sulfate (pH 6) was obtained from Sigma. The ammonium sulfate was removed by dialyzing twice against deionized water (6 h each) and then against 20 mM potassium phosphate at pH 7 (12 h). The ANC solution was centrifuged and the deep green solution was chromatographed using the same procedure described for BLC. The purified ANC solution (Rz = 0.68) was used for the spectroscopic measurements. The buffer exchange was achieved either by passing the catalase solution over a Sephadex G-25 (Pharmacia) column or by ultrafiltration (Amicon). The buffers used for the final samples were 250 mM sodium acetate (pH 5), 250 mM potassium phosphate (pH 7), and 250 mM sodium carbonate (pH 10.5 and 11.6).

Potassium cyanide was from Aldrich. The KCN, KCN, and KCN at 99% enrichment were obtained from Isotech Inc. (Miamisburg, OH). The various isotopomeric forms of cyanoferric ANC and BLC were prepared by mixing 0.15 ml of catalase solution (approximately 0.1 mM in heme) with 0.05 ml of the isotopomeric cyanide solution (160 mM).

Spectroscopic Measurements

The RR spectra were acquired with a Spex 1269 spectrometer (mechanical slit width of 75 µm) equipped with a Princeton Instruments ICCD-576 uv-enhanced detector and a 413.1-nm notch filter (Kaiser, Ann Arbor, MI). The 413.1-nm laser line was from a Coherent Innova model 100-K3 Krypton ion laser. The spectra were collected with the back scattering geometry from 5-mm NMR tubes. The NMR tube containing the sample was spun during illumination to avoid local heating. In all cases a gradually increasing background was observed which became more serious at high pH values, especially for BLC. The frequencies of the severely overlapping isotope-sensitive features were estimated using the comparison of the difference spectra obtained by subtraction of absolute experimental spectra for different isotopes with computer-simulated difference spectra (see below).

Computer Simulations

To obtain a more reliable estimate of the actual vibrational parameters of individual modes from the difference spectra, the following approach has been used. Initially, the idealized (built of ideal, 50/50, Gauss-Lorentzian peaks) absolute spectra are simulated by computer. These simulated spectra are then subtracted and the difference spectra obtained in this way are compared with their experimental analogues. In the next step the idealized absolute spectra are slightly perturbed (by allowing frequency, band width, and intensity of peaks to vary) and the new set of simulated difference spectra is compared with the set of experimental difference spectra. The perturbation process is repeated iteratively until the discrepancy between the simulated and experimental difference spectra is minimized. The computer program based on this strategy has been locally generated.() In this program the quantitative measure of the difference between the simulated and the experimental data ( D) is defined by the following equation.

On-line formulae not verified for accuracy

The results corresponding to the best fit for ANC-CN at pH = 4.9 and BLC-CN at pH 7.0, obtained by the computer program described above, are summarized in . In the process of simulation it has been assumed that different isotopic substitutions in the CN fragment influence the absolute frequencies of the peaks only and that the band widths and intensities do not change upon these substitutions. Various initial estimates and perturbation steps have been used in the process of fitting. In all cases the frequencies obtained were within experimental error ± 2 cm of those summarized in .


RESULTS

Identification of the Fe-C-N Axial Modes

BLC-CN

Fig. 1 shows the RR spectra of the various isotopomeric forms of BLC-CN. To help identify the Fe-C-N modes, we digitally generated difference spectra (Fig. 2) by the subtraction of the various pairs of spectra shown in Fig. 1. Inasmuch as the Fe-C-N axial modes overlap, and the difference features in Fig. 2therefore do not necessarily correspond to the absolute frequencies, we have developed a simulation procedure described above which allows us to estimate the absolute frequencies of the peaks contributing to the experimental difference spectra. Fig. 3shows the results of computer simulation of experimental difference spectra. The peak positions estimated from the fitting of the difference spectra between 340 and 460 cm for the CN adducts of BLC are summarized in .() An easily identified isotope-sensitive mode in the spectrum of natural abundance BLC-CN can be seen at 433 cm in Fig. 1 , a mode that is absent in the RR spectra of the native BLC. This mode exhibits a monotonic isotope shift pattern (Yu et al., 1984) as the total mass of the cyanide isotopomer is increased, shifting to 431 cm (CN), 430 cm (CN), and 429 cm (CN). A feature that corresponds to this mode can be seen in Fig. 2. On the basis of this isotope shift pattern we assign this mode to the [Fe-CN] stretching vibration in BLC-CN (Yu et al., 1984). In Fig. 1, there is an apparent isotope-sensitive intensity fluctuation observed in the region between 400 and 416 cm. Upon labeling the carbon atom of the bound cyanide, the mode at 416 cm decreases in intensity and shifts 4-5 cm to higher frequency. Simultaneously, a shoulder at 404 cm appears only when the carbon atom of the bound cyanide is labeled. The digitally subtracted difference spectra, given in Fig. 2 , clearly document the presence of this isotope-sensitive mode. Traces `` c-b,'' ``a-b,'' ``a-d,'' and `` c-d'' of Fig. 2, which are obtained by the subtraction of spectra of BLC-CN isotopomers in which the carbon atom of the ligated cyanide is labeled from spectra of cyanide adducts in which the carbon atom is not labeled, show difference features at 412 and 402 cm. This mode cancels out completely in traces ``a-c'' and ``b-d,'' which are obtained by the subtraction of a pair of spectra corresponding to the isotopomers in which the carbon atoms have the equal masses. As is clear from inspection of Figs. 2 and 3 this behavior results from the presence of a band which exhibits a zigzag isotopic shift pattern, shifting (based on the simulation) from 413 cm (CN), to 402 cm (CN), 412 cm (CN), and 401 cm (CN). Accordingly, we assign this mode to a [Fe-C-N] bending vibration (Yu et al., 1984).


Figure 1: Low frequency RR spectra of the different isotopomeric forms of the cyanide adducts of bovine liver catalase at pH 7.




Figure 2: Difference spectra generated by the digital subtraction of the various spectra shown in Fig. 1.




Figure 3: Difference spectra of the BLC-CN at pH 7 generated by the simulation procedure.



Recently, we reported that cyanide binds to ferric HRP in two geometries (Al-Mustafa and Kincaid, 1993). Therefore, we examined the spectra for evidence of heterogeneity in binding of cyanide to ferric BLC. Fig. 2reveals the presence of an isotope-sensitive mode hidden by the strong porphyrin mode () at 349 cm. This mode can be seen in all traces of Fig. 2with the exception of trace c-b. These difference spectra were generated by the subtraction of spectra of isotopomeric forms which differ in the total mass of the ligated cyanide, while it cancels out completely in trace c-b, which was obtained by the subtraction of the spectrum of the CN adduct from that of the CN adduct ( i.e. those which have equal total masses). These data thus document the presence of a mode which shifts monotonically with increase of the total mass of the cyanide ligand, regardless of the position of labeling (Yu et al., 1984). The overlap of this band with an intense porphyrin mode () hinders the determination of the exact frequency of this mode. However, with the help of the simulation procedure and using the difference spectra shown in Fig. 2 , we estimate that this mode occurs at 349 cm for the CN adduct and shifts to 345 cm for the CN adduct. Accordingly, we assign this mode to the [Fe-CN] stretching mode of a second conformer of BLC-CN (Han et al., 1989; Lopez-Garriga et al., 1990; Hu et al., 1993; Al-Mustafa and Kincaid, 1993).

The presence of a fourth isotope-sensitive mode ( i.e. a second bending mode) is less apparent in both the absolute and the difference spectra shown in Figs. 1 and 2, respectively. However, careful examination of the difference spectra in Fig. 2 , especially traces a-b, a-d, and c-d, which are expected to show all of the stretching and bending modes, reveals an asymmetrical difference feature near 430 cm. On the other hand, traces a-c and b-d of Fig. 2, which should show only the stretching modes (the bending modes should cancel out) yield a symmetrical difference feature in the same region. Therefore, we conclude that the asymmetry of the difference features in traces a-b, a-d, and c-d of Fig. 2, in the region around 430 cm, is due to the combination of two overlapping difference features; a feature that is ascribable to the monotonically shifting mode at 433 cm mentioned earlier and assigned to a stretching of [Fe-CN], and a second mode of slightly higher frequency that exhibits a zigzag isotopic shift pattern. In trace c-b of Fig. 2, which is obtained by the subtraction of isotopomeric forms that have equal total masses but differ in the mass of the carbon atoms, should reveal only the bending vibration ( i.e. the stretching vibrations should cancel out). This trace clearly reveals the presence of two bending vibrations; the feature near 410 cm mentioned earlier, and another bending mode near 440 cm. The numerical fitting yielded two simulated bands, having a zigzag pattern, in these regions. One mode at 413 cm (CN) shifting to 401 cm (CN) and a second mode at 445 cm (CN) that shifts to 435 cm (CN).

Studies carried out on the cyanide adducts of the one electron-reduced sulfite reductase (Han et al., 1989), ferric HRP (Al-Mustafa and Kincaid, 1993), ferric myeloperoxidase (Lopez-Garriga et al., 1990), and ferric lactoperoxidase (Hu et al., 1993) revealed that the bending of the Fe-C-N linkage causes a reversal of the ordering of the [Fe-CN] and [Fe-C-N] vibrations and drives them apart relative to the linear form. Therefore, we assign the stretching mode at 434 cm and the bending mode at 413 cm to a conformer of ferric BLC-CN which possesses an essentially linear Fe-C-N linkage and the stretching and the bending modes at 349 cm and 445 cm, respectively, to a second conformer having a more bent Fe-C-N linkage.

Inspection of Figs. 1 and 2 reveals the presence of several other isotope-sensitive features which are not readily discernable in Fig. 1owing to their small shift and their overlap with isotope-insensitive heme deformation modes. The region near 380 cm in Fig. 1 appears to be composed of two modes; one of which is a mode at 380 cm which exhibits a small (2 cm) but definite downshift. The observation of sharp difference features near 380 cm in the difference spectra in Fig. 2confirms the isotope sensitivity of this mode. The behavior of this component upon isotopic substitution is similar to a mode observed at 378 cm in the RR spectra of cyanide adducts of HRP (Al-Mustafa and Kincaid, 1993). We assign this vibration, as it was assigned in the case of HRP-CN, to an out-of-plane heme core mode or possibly a peripheral substituent mode and we attribute the observed shifts to the coupling of this mode to the Fe-C-N vibrations, which are also out-of-plane in nature. The second component can be seen as a shoulder at 385 cm which does not show any isotope dependence.

ANC-CN

The RR spectra of ferric ANC-CN obtained at pH 4.9 and their difference spectra are shown in Figs. 4 and 5, respectively. Fig. 4shows an isotope-sensitive mode at 433 cm (CN), 432 cm (CN), 431 cm (CN), and 429 cm (CN). This band corresponds to the 434 cm mode observed for BLC-CN and accordingly is assigned also to the [ Fe-CN] mode of a linear conformer of ANC-CN. A second isotope-sensitive mode that exhibits a zigzag shift pattern can be seen near 410 cm in both Figs. 4 and 5. Similarly to BLC-CN, simulations suggest that this mode occurs at 412 cm (CN) and at 402 cm (CN) and is assigned to the [Fe-C-N] bending vibration of the linear conformer of ANC-CN.


Figure 4: Low frequency RR spectra of the different isotopomeric forms of the cyanide adducts of A. niger catalase at pH 4.9.



As in the case of BLC-CN, the spectra of ANC-CN reveal the presence a second conformer with a reversed ordering of the stretching and bending frequencies. The difference spectra in Fig. 5 reveal the presence of a monotonically shifting mode near 350 cm (CN), shifting to 347 cm (CN) and it is assigned to the [Fe-CN] stretching vibration of a second, bent, conformer of ANC-CN. The major difference between the spectra of BLC-CN and ANC-CN is the frequency of the bending mode identified at 445 cm in BLC-CN. This mode appears to shift to a slightly higher frequency in ANC-CN as is indicated by the appearance of a new positive component at about 456 cm in traces a-b, a-d, and c-d of Fig. 5 . Also trace c-b of Fig. 5, which is expected to exhibit only the difference features associated with the bending vibrations of ferric ANC-CN, shows a difference feature at 455 and 443 cm slightly higher than BLC-CN (444 and 436 cm). The absolute spectra of ANC-CN in Fig. 4 show a weak feature at 456 cm (CN, CN) that apparently disappears, most likely shifting underneath the strong band at 431-429 cm(CN, CN). Accordingly, it is assigned to the [Fe-C-N] bending vibration of the second form of ferric ANC-CN. We assign the features at 350 and 456 cm to the stretching and bending vibrations of a second conformer of ANC-CN which exists in a bent Fe-C-N geometry. The results of simulation (Fig. 6) of the experimental difference spectra suggest another significant difference in the spectral behavior of BLC-CN and ANC-CN besides the upshifting of the bending mode from 445 cm to 456 cm; i.e. the isotope shift upon C substitution for the [Fe-C-N] of the bent conformer has been found to be 10 cm for BLC-CN and 15 cm in the case of ANC-CN. The 10 cm isotopomeric shift found in the case of BLC-CN is in reasonable agreement with the experimental data reported for related systems, where the shift varied between 5 and 8 cm (Lopez-Garriga et al., 1990; Han et al., 1989; Hu et al., 1993). pH Effect


Figure 5: Difference spectra generated by digital subtraction of the various spectra in Fig. 4.




Figure 6: Difference spectra of ANC-CN at pH 4.9 generated by the simulation procedure.



In order to investigate the hydrogen bonding interactions of the bound cyanide with the active site distal environment, we have studied the RR spectra of BLC and ANC in the pH range 4.9-11.5. Analysis of the high and low frequency RR spectra of the native form (spectra not shown) and the cyanide-ligated forms indicates no significant denaturation for either protein. However, at pH values slightly below 5, BLC starts to precipitate while ANC remains soluble (possibly because ANC is a glycoprotein containing about 9% carbohydrate while BLC does not contain any carbohydrates) (Kikuchi-Torii et al., 1982). The fluorescence from the catalase samples increases significantly as the pH of the medium is raised above 10, especially for BLC. Nevertheless RR of adequate quality spectra can be obtained at pH values as high as 11.5.

Fig. 7 shows the low frequency RR of the isotopomeric CN adducts of BLC at pH 11.5. The most noticeable changes in the frequencies of the axial modes is in the [Fe-CN] stretching frequency of the linear conformer. The feature which occurs at 433 cm at pH 7.0 (Fig. 1) shifts to 429 cm at pH 11.5. Therefore, we assign the feature at 429 cm to the [Fe-CN] mode of the alkaline form of the linear conformer. It is clear that changing the pH in the range 4.9-11.5 does not eliminate the heterogeneity as indicated by the observation of two difference features near 430 and 350 cm in the difference traces shown in Fig. 8. Two additional minor downshifts can be noticed comparing the difference traces at pH 11.5 (Fig. 8) to the traces at pH 7.0 (Fig. 2). One is the small 2-4 cm downshift of the positive feature at about 440 cm and the second is the 4 cm downshift of the negative feature at about 400 cm.


Figure 7: Low frequency RR spectra of the ANC-CN adducts at pH 11.5 in 250 mM carbonate buffer.




Figure 8: Difference spectra generated by the digital subtraction of the various spectra shown in Fig. 7.



RR investigation of the cyanide adducts of ferric ANC at pH 11.5 (not shown) yield spectra practically identical to those obtained at pH 4.9. Also the difference spectra (Fig. 9) show no observable differences compared to traces obtained at pH 4.9 (Fig. 5). Thus, we detect no significant shifts in axial vibrational modes of ANC-CN in the pH range 4.9-11.5.


Figure 9: Difference spectra generated by the digital subtraction of the low frequency RR spectra (not shown) of the different isotopomeric forms of ANC-CN adducts at pH 11.5.



The absolute frequencies for the CN adducts of both BLC-CN and ANC-CN, at both high and low pH, are summarized in . The values of frequencies, band widths, and relative intensities at low pH for both adducts have been obtained from the computer simulation of the experimental difference spectra. The frequencies shown for both BLC-CN and ANC-CN at pH 11.5 have been estimated by comparing their difference traces with corresponding low pH analogue and using the information from the low pH simulation data.


DISCUSSION

Spectral Behavior

The RR data presented for BLC-CN and ANC-CN unambiguously demonstrate the presence two conformers characterized by the reversal of the ordering of their [Fe-CN] stretching and [Fe-C-N] bending frequencies. In the first conformer, the observed frequency of [Fe-CN] (434 cm) is higher than the [Fe-C-N] (413 cm) frequency. This behavior has been observed previously for the cyanide adducts of several heme proteins and model compounds (Yu et al., 1984; Yu and Kerr, 1988; Han et al., 1989; Lopez-Garriga et al., 1990; Tanaka et al., 1987; Uno et al., 1988; Al-Mustafa and Kincaid, 1993) and is considered to be indicative of an essentially linear Fe-C-N linkage. In the second conformer the [Fe-CN] mode (349 cm) occurs at a lower frequency than the [Fe-C-N] mode (445 cm for BLC-CN and 456 cm for ANC-CN), behavior which also has been observed in the cyanide adducts of several heme proteins ( e.g. myeloperoxidase (Lopez-Garriga et al., 1990), lactoperoxidase (Hu et al., 1993), sulfite reductase (Han et al., 1989), and HRP (Al-Mustafa and Kincaid, 1993)) and is characteristic of a nonlinear Fe-C-N linkage. The essential from all of these previous studies are summarized in .

Structural Interpretation

The crystal structures of BLC, PVC, and MLC (Murthy et al., 1981; Vainshtein et al., 1986; Murshudov et al., 1992) reveal the presence of a conserved essential histidine residue on the distal-side of the heme active sites. Furthermore, kinetic studies of cyanide binding (Chance, 1952; Schonbaum and Chance, 1976) indicate that catalases bind HCN rather than CN with the proton presumably protonating the distal histidine, as in the case of HRP and cytochrome c-peroxidase (Thanabal et al., 1988; Banci et al., 1991). The observed heterogeneity can reasonably be attributed to distinct hydrogen bonding interactions between the Fe-C-N linkage and two different distal-side amino acid residues. Thus, the bound cyanide can participate in a hydrogen bonding interaction with the imidazolium group of the distal histidine, giving rise to a linear conformer, which exhibits the characteristic stretching and bending vibrations (434 and 413 cm, respectively). The bent conformer presumably arises from an off-axis hydrogen bonding interaction with another residue. Apparently because of slight differences in the distal-side environment, the hydrogen bonding interactions which give rise to the bent conformers of BLC-CN and ANC-CN appear to be somewhat different as indicated by the variation in the [Fe-C-N] frequency of the bent conformer (observed at 445 and 456 cm for BLC-CN and ANC-CN, respectively) and the increased isotopic shift for ANC relative to BLC. The 10 cm upshift in the bending frequency of the bent conformer of ANC-CN relative to that of BLC-CN can be attributed to variation in binding geometry of the cyanide or in the strength of the hydrogen bonding interaction with the heme distal environment. This difference in behavior between the cyanide adducts of ANC and BLC is consistent with the known structural differences in heme distal between mammalian and fungal catalases (Murthy et al., 1981; Vainshtein et al., 1986) as is discussed below.

In addition to the distal histidine (His-74), the crystal structure of BLC reveals the presence of a distal asparagine residue (Asn-147), an amino acid known to be capable of acting as a hydrogen bond donor to the bound cyanide (Murthy et al., 1981; Fita and Rossman, 1985a). Therefore, it is reasonable to suggest that a hydrogen bonding interaction of the bound cyanide with the distal asparagine (known to be displaced further from the heme normal than the histidine) produces the bent Fe-C-N linkage which exhibits its characteristic [Fe-CN] and [Fe-C-N] frequencies (350 and 445 cm, respectively). Comparison of the available crystal structures of the various catalases reveals a high degree of similarity in their heme distal environments (Murthy et al., 1981; Vainshtein et al., 1986). The asparagine residue (Asn-147) found in BLC (Murthy, 1981) is conserved in both PVC (Asn-132) (Vainshtein et al., 1986) and MLC (Asn-129) (Murshudove et al., 1992).

pH Effects

It is generally agreed that the distal-side of the heme active sites of catalases and peroxidases contain two or more amino acids capable of participating in hydrogen bonding interactions with the natural substrate, hydrogen peroxide (Finzel et al., 1984; Fita and Rossman, 1985a; Sakurada et al., 1986; Thanabal et al., 1988). Furthermore, it has been suggested that these residues promote the heterolysis of the O-O bond of HO and the formation of the reactive intermediate by stabilizing the developing charge separation and facilitating the proton transfer to the terminal oxygen of HO (Finzel et al., 1984; Fita and Rossman, 1985a).

The presence of hydrogen bonding between these amino acid residues and the bound cyanide may impart sensitivity of the Fe-C-N vibrations to changes in pH, since raising the pH above the p Kof the amino acid residue would eliminate the hydrogen bond and consequently decrease the axial mode frequencies (Lopez-Garriga et al., 1990; Hu et al., 1993; Al-Mustafa and Kincaid, 1993). Comparison of Figs. 1 ( trace a) and 7 shows that raising the pH from 7 to 11.5 causes a 4 cm downshift in the [Fe-CN] stretching frequency of the linear conformer of BLC-CN. This response to pH changes is analogous to the downshift observed for the linear conformer of cyanoferric HRP, and can reasonably be ascribed to the deprotonation of the imidazolium group of the distal histidine (Al-Mustafa and Kincaid, 1993). In contrast to the behavior observed for cyanoferric HRP, the [Fe-CN] vibration of the bent conformer does not show any significant pH-dependent shift in this pH range. This behavior is not only consistent with hydrogen bonding interaction with the distal asparagine (which is not deprotonated at this pH), but also indicates that the distal- and the proximal-sides of the heme active site in catalases function independently. This behavior is different from that observed in HRP and other peroxidases wherein deprotonation of the distal histidine causes a corresponding change in the trans-axial bond strength (Teroaka and Kitagawa, 1981).

RR investigation of the ANC-CN in the pH range 4.9-11.5 did not reveal any significant shift in the axial mode frequencies of either the linear or the bent conformers (Figs. 5 and 9). The lack of a pH-dependent shift in ANC-CN presumably reflects a greater stability of the hydrogen bond between the distal histidine and the bound cyanide, although no structural argument for this increased stability can be made in the absence of definitive active site structural data.

Comparison with Cyanide Adducts of HRP and Other Heme Proteins

Recently we reported the results of a RR investigation of the cyanide adducts of HRP (Al-Mustafa and Kincaid, 1993). Although HRP and the two catalases studied in this report exhibit evidence for the existence of two conformers (linear and bent), several significant differences can be noted. First, the [Fe-CN] stretching vibration (434 cm) of the linear conformers of catalases are considerably lower than that observed for the linear conformer of HRP and other heme proteins that bind cyanide in an essentially linear configuration (456 cm) (). In fact, this is the lowest reported [Fe-CN] stretching frequency identified for a linear heme protein cyanide adduct. Also the [Fe-CN] modes of the bent conformer of both BLC-CN and ANC-CN (about 350 cm) are 10 cm lower than that of HRP-CN (360 cm) and are virtually identical to that of the bent conformer of the one-electron reduced sulfite reductase (352 cm) which also possesses a negatively charged proximal ligand ( i.e. thiolate) (Han et al., 1989). Second, the magnitudes of the observed isotope-dependent shifts of stretching modes for linear conformers of the catalases are apparently slightly smaller than that observed for the linear conformer of HRP (substitution of CN for CN yields 4-6 cm shifts for the catalases but an 8 cm shift for HRP-CN). Third, we reported for HRP-CN that the deprotonation of a single amino acid leads to downshifts of the [Fe-CN] for both the linear conformer (attributed to the direct effect of abolishing the hydrogen bond with bound cyanide) and for the bent conformer (through the indirect alteration of the proximal Fe-N(His) bond strength) (Al-Mustafa and Kincaid, 1993). However, in BLC-CN only the [Fe-CN] mode of the linear conformer downshifts upon raising the pH while that of the bent form remains unchanged.

These differences in behavior between catalase and HRP can be reasonably explained in terms of the variation in active site environments between catalase and HRP. Many factors might contribute to this unusually low [Fe-CN] stretching vibration ( e.g. axial ligation, hydrogen bonding, and the variation in the degree of tilting or bending). A major factor that might contribute to the lowering of the [Fe-CN] is the unique distal-side structure of the heme active site in catalase. The crystal structures of the three catalases (Murthy et al., 1981; Vainshtein et al., 1986; Murshudov et al., 1992; Fita and Rossman, 1985a) reveal that the essential distal histidine (Margoliash et al., 1971) is almost parallel to the heme plane. This orientation of the distal histidine is different from the orientation found in cytochrome c-peroxidase (Finzel et al., 1984) and that expected for HRP (Sakurada et al., 1986). Thus, the steric and hydrogen bonding interactions between the bound cyanide and the distal histidine in catalases are expected to increase the tension on the Fe-C-N linkage, increasing the off-axis distortion relative to that of HRP. This should lead to further lowering of the [Fe-CN] stretching frequency, and to the observed decrease in the magnitude of the isotopic shift in catalase relative to those observed for the cyanide adducts of HRP and sulfite reductase (Al-Mustafa and Kincaid, 1993; Han et al., 1989). Another distinctive feature of the catalase active site structures is the coordination of a phenolate oxygen from an endogenous tyrosine residue to the heme iron (Murthy et al., 1981; Vainshtein et al., 1986; Murshudov et al., 1992). It has been shown that increasing the basicity of the trans-axial ligand strength in the cyanide-ligated FeOEP complexes leads to a corresponding decrease in the [Fe-CN] stretching frequency (Uno et al., 1988). Similar behavior has been detected for the CO adducts of model compounds, in studies which demonstrate that the [Fe-CO] frequency is highest when the trans-axial ligand is very weak or absent (Kerr et al., 1983). Recently, for the CO adducts of BLC, we reported (Hu and Kincaid, 1992) the observation of the [Fe-CO] and the [C-O] at 542 and 1908 cm, respectively. These values are comparable to those reported for the CO adducts of HRP (537 and 1904 cm, respectively (Evangelista-Kirkup et al., 1986) and 541 and 1906 cm (Uno et al., 1987)). Furthermore, the reported [Fe-CO] and [ C-O] values correlate with those of other heme proteins and model compounds containing a nitrogenous trans-axial ligand, indicating that the -donating effect of the proximal phenolate oxygen in catalase is comparable to that of the imidazole found in HRP and other heme proteins (Yu et al., 1988; Li and Spiro, 1988). Therefore, the difference in the proximal ligands between catalase and HRP is not expected to contribute significantly to the lowering of the [Fe-CN] frequencies of catalases.

The difference in response to pH changes between catalase and HRP is also consistent with active site structural variations between the two proteins. It is generally agreed that deprotonation of the distal histidine in HRP produces a corresponding alteration of the trans Fe-N(His) bond strength (Teroaka and Kitagawa, 1981; Thanabal et al., 1988). The 4 cm pH-induced shift observed for the [Fe-CN] mode of the bent conformer of HRP-CN is attributable to a change in the trans-axial ligand bond strength which is triggered by the deprotonation of the distal histidine (Teroaka and Kitagawa, 1981). The lack of any measurable shift in the [Fe-CN] mode of the bent conformer of BLC-CN implies that the distal- and the proximal-sides in catalase function independently.

  
Table: Axial mode frequencies (cm), band widths (FWHM in cm) and relative intensities obtained by computer simulation of difference spectra of cyanoferric HRP with different labeled cyanide isotopomers


  
Table: The comparison of cyanide adducts of various heme proteins

The following abbreviations are used: BLC, bovine liver catalase; ANC, Aspergillus niger catalase; HRP, horseradish peroxidase; SiR-HP, E. coli sulfite reductase in its oxidized form; MetHb CTT, hemoglobin III from Chironomus thumni thumni; LPO, lactoperoxidase; MPO, myeloperoxidase, CCP, cytochrome c peroxidase.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 35153. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: BLC, beef liver catalase; PVC, Penicillium vitale catalase; MLC, Micrococcus lysodeikticus catalase; HRP, horseradish peroxidase; ANC, Aspergillus niger catalase; RR, resonance Raman.

M. Sykora and J. R. Kincaid, manuscript in preparation.

Together with estimation for the isotope-sensitive porphyrin mode.


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