NO stretching, Fe(III) - Semantic Scholar

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Aug 1, 1983 - Champion, P. M., Stallard, B. R., Wagner, G. C. & Gunsalus, I. .... 385. 42. Irwin, M. J., Armstrong, R. S. & Wright, P. E. (1981) FEBS Lett.

Proc. Nati. Acad. Scd USA Vol. 80, pp. 7042-7046, November 1983 Chemistry

Resonance Raman studies of nitric oxide binding to ferric and ferrous hemoproteins: Detection of Fe(III)-NO stretching, Fe(III)-N-O beding, and Fe(II)-N-O bending vibrations (myoglobin/hemoglobin/horseradish peroxidase/ligand vibrations)

B. BENKOt AND NAI-TENG Yut School of Chemistry, Georgia Institute of Technology, Atlanta, GA 30332

Communicated by J. L. Oncley, August 1, 1983

ABSTRACT The nature of bonding interactions between Fe(EI) and NO in the ferric nitrosyl complexes of myoglobin (Mb), hemoglobin A (HbA), and horseradish peroxidase (HRP) is investigated by Soret-excited resonance Raman spectroscopy. On the basis of 5NO and N'80 isotope shifts, we clearly identified the Fe(IE)-NO bond stretching frequencies at 595 cm-1 (ferric Mb'NO), 594 cm- (ferric HbA'NO), and 604 cm- (ferric HRP-NO). The Fe(IEi)-N-O bending vibrations are located at 573 cm-' (ferric Mb'NO) and 574 cm-' (ferric HRP'NO), which are very similar to the Fe(l)-C-O bending modes at 578 cm 1 in Mb'CO and HbA-CO. However, the Fe(III)-NO and Fe(II)-CO stretching frequencies differ by -90 cm-f, indicating a much stronger iron-axial ligand bond for the [Fe(Iil) + NO] system, which is isoelectronic with the [Fe(Il) + CO] system and, hence, presumably also has a linear Fe(HI)-N-O linkage (in the absence of distal steric effect). The unusually strong Fe(IH)-NO bond may be attributed to the ir bondin involving the unpaired electron in the ir*(NO) orbital. The N1 0 isotope shift data indicate that the widely accepted assignment of the Fe(II)-NO stretching vibration at "554 cm-' in ferrous nitrosyl Mb/HbA is incorrect; instead, we assign it to the Fe(il)-N-O bending mode. The validity of the assignment of Fe(i)-02 stretch at 567 cmin oxy-HbA by Brunner [Brunner, H. (1974) Naturwissenschaften 61, 129-130] is now in doubt. Literature data are presented to suggest that it is the Fe(Il)--O-O bending vibration.

ing mode, investigators often assigned the only isotope-sensitive line in the 100- to 700-cm-1 region as the iron-ligand stretching mode. However, it has been demonstrated (10, 11) that, in azide complexes of metmyoglobin, methemoglobin, and manganese-substituted myoglobin, only ligand bending modes were enhanced when the metal-ligand stretching modes were not detectable. Nitric oxide binds to both ferric and ferrous hemoproteins (12-20). Although ferrous nitrosyl complexes have been extensively studied by x-ray diffraction (21-24), infrared (24, 25), visible absorption (26-28), EPR (19, 20, 22, 29, 30), extended xray absorption fine structure (EXAFS) (31), and resonance Raman spectroscopy (8, 32-39), very little is known regarding the bonding interactions between Fe(III) and NO in hemoproteins and heme model compounds, presumably because of the intrinsic tendency towards spontaneous autoreduction (12-20), the diamagnetic property (EPR silent) (14, 15), and the high quantum yield of ligand photodissociation (40) have hindered meaningful resonance Raman studies. In this paper, we demonstrate the feasibility of obtaining highquality resonance Raman spectra of nitrosyl complexes of Mb, hemoglobin A (HbA), and horseradish peroxidase (HRP) in both ferric and ferrous forms. The Fe(III)-NO stretch has been clearly identified (by using '5NO and N180 isotopes) at -600 cml, which is much higher than the Fe(II-CO stretch at =500 cm-' (4), the Fe(III)-S stretch at =350 cm-' (5), and the Fe(III)-CN- stretch at 454 cm-' (unpublished results). The Fe(III)-N--O bending vibration is assigned to the =573-cm 1 line on the basis of its "zigzag" pattern. We were surprised to find that a similar zigzag pattern exists with the 554-cm-1 line in ferrous nitrosyl Hb/Mb; this line was previously assigned to the Fe(II)-NO stretch by Chottard and Mansuy (8). Here, we assign it to the Fe(II)-N--O bending mode. The generally accepted assignment of the Fe(II)-02 stretch at =570 cm-1 in oxy-Hb/Mb (1-4, 7-9, 32, 37, 39, 41-46) is reassessed by using the Raman data from oxy-Hb with 16018O isotope (46); it is suggested as the Fe(II)-O--O bending mode.

Resonance Raman spectroscopy is an ideal tool for directly monitoring the iron-axial ligand bond strength in hemoproteins and synthetic model hemes (refs. 1-3 and references cited therein). The information it provides is important in understanding the mechanisms of protein control of heme reactivity. At present, there are several stretching vibrations of iron-axial ligand bonds that have been unambiguously identified. For example, the Fe-C bond in (carbonmonoxy) hemoglobin (Hb) and myoglobin (Mb) (4), Fe-S bond in cytochrome P450 (camphor) (5), Fe-Ne (proximal histidine) bond in deoxy-Hb/Mb (6, 7), and Fe-CN bond in cyanometerythrocruorin (unpublished results). The assignments of Raman lines characteristic of these vibrations were made through at least two different isotope shifts. However, there are important assignments such as the Fe(II)-NO stretch in Hb-NO (8) and the Fe(II)-02 stretch in Hb-02 (9) that were based on a single isotope shift. Such an experimental approach does not enable one to distinguish between iron-ligand stretching and bending vibrations. On the basis of a tacit assumption that the iron-ligand stretching vibration should be more readily enhanced than the bend-

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MATERIALS AND METHODS Sperm whale Mb (Sigma) was purified in the ferric form by the procedure described previously (11). Human hemoglobin Ao (HbA) was prepared in the oxy form from whole blood as described by Huisman and Dozy (47), and was converted to the Abbreviations: HRP, horseradish peroxidase; HbA, hemoglobin A; Mb,

myoglobin. t Present address: Institute of Immunology, Rockefellerova 2, Zagreb, Yugoslavia. * To whom reprints requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7042

Chemistry: Benko and Yu ferric form by oxidation with potassium ferricyanide, which was subsequently removed by extensive dialysis against 0.05 M sodium phosphate buffer at pH 6.0. HRP from Worthington (A4M/ A275 = 3.16; activity = 1,058 units/mg) was purified on Sephadex A-50 and CM-cellulose column according to Paul and Stigbrand (48); only C type HPR was used in this study. Heme concentrations were determined spectrophotometrically, with the following extinction coefficients: 11.0 mM'1 cm-' at 540 nm (ferric MbCN- and ferric HbCN-) (49) and 100 mM-1 cm-1 at 403 nm (resting HRP) (48). For the time-dependence study of the autoreduction of ferric Mb-NO or ferric HbA-NO, the sample solution (in ferric form) was diluted to the desired concentration (60-90 ,uM) with appropriate buffer and then transferred to a Raman cell fitted with a rubber septum. The oxygen in the solution was removed by repeated evacuation and flushing with pure nitrogen gas (Matheson, CP grade). After the final evacuation, nitric oxide (Matheson, CP grade) washed with concentrated NaOH solution (Q0. 1 M) was introduced at a pressure of %z1 atmosphere (101 kPa) or slightly less. To study ferric Mb-NO with different isotopes (14N160, 15N'60, and 14N'80), Raman spectra were recorded within less than 7 min after the mixing of ferric Mb solution with NO gas. After =8 hr of standing at room temperature, the sample of ferric Mb-NO (or ferric HbA-NO) was spontaneously converted to ferrous Mb-NO, without addition of sodium dithionite (12-15). For control experiments the ferric form of Mb (or HbA) was anaerobically reduced in the Raman cell by injecting a slight excess of sodium dithionite solution (buffered and degassed) before nitric oxide was introduced. The two methods produced identical resonance Raman spectra of ferrous Mb-NO (or HbANO). The samples of nitric oxide composed of different isotopes were obtained from the following sources: 15NO (Stohler Isotope Chemicals, Waltham, MA; 99% enrichment in '5N) and N'80 (custom-synthesized by Prochem US Services, Summit, NJ; 90% enrichment in 180). Resonance Raman spectra were recorded with a highly sensitive multichannel system (S0) consisting of a modified Spex 1402 0.85-m Czerny-Turner double monochromator (two 600 grooves per mm gratings in additive dispersion), a dry ice-cooled (-620C) intensified vidicon detector (Princeton Applied Research model 1254), a PAR 1216 detector controller, a Tektronix 604 monitor, and a PAR model OMA 2 microprocessor-based console. Light at the excitation wavelengths 406-.7 and 413.1 nm was provided by a Spectra-Physics model 171 Kr' laser. The ligand photodissociation, especially the ferric Mb-NO having a quantum yield of -1 (40), was minimized by spinning the Raman cell (=2,000 rpm) during laser irradiation. The slit width used was 100 jAm and the slit height was 2 mm. Spectra were calibrated by using fenchone as a standard compound (50). Additional standards used for the high-frequency region were toluene (1379, 1385, and 1604 cm-') or benzene (1585 and 1606 cm'1). The Raman spectra presented here have not been computer-smoothed. Reported wavenumbers are accurate within ±1 cm-1 for sharp lines and ± 2 cm-1 for broad lines. RESULTS Resonance Raman spectra (200-650 cm-') of ferric Mb-NO with various isotopes are presented in Fig. 1. Spectra were obtained within 7 min after the addition of NO; there is no significant amount of ferrous species present, as judged by the absence of a distinct line at 357 cm-'. There were virtually no changes in the spectrum when the laser power was increased from 5 to 30 mW, nor were any changes observed when the pH was varied between 8.4 and 5.8. There are two isotope-sensitive lines at

Proc. Natl. Acad. Sci. USA 80 (1983)

200 300 400

7043

500 600

Frequency (cm-1)

FIG. 1. NO isotope effects on resonance Raman spectra (200-650

cmn1) of ferric Mb-NO. Conditions: excitation A, 406.7 nm; power, 15

mW; delayed cycles, 1,000 (30.3 sec); slit width, 100 pam; slit height, 0.2 cm; concentration, 76 ,M in 0.05 M Tris-HCl, pH 7.2.

595 and 573 cm-1. The 595-cm-1 line shifts to 589 (15NO) and 587 cm-' (N'80) as the NO mass increases by 1 and 2 daltons, respectively. In contrast, the 573-cm-1 line decreases to 562 cm-' (5NO), which then increases to 569 cm-1 (N180). On the basis of the observed shift from NO to 15NO, one would expect the frequency to decrease again from N'80 to 15N180. The remaining features in the 200- to 650-cm-1 region are typical of ferric Mb derivatives such as aquo-, fluoro-, azido-, and hydroxyl-met-Mb (unpublished results). The 411-cm-1 line may be assigned to the vinyl group bending mode on the basis of recent work by Choi et al. (51) and Rousseau et al. (52). On standing for =7 hr or longer, the ferric Mb-NO complex underwent autoreduction and was converted completely to the ferrous Mb-NO species. The resulting spectra with NO and "5NO are identical to those reported recently from this laboratory (38). Contrary to the work of Walters and Spiro (39), we found no pH dependence of ferrous Mb-NO spectrum between 8.4 and 5.8. The resonance Raman spectrum of ferrous Mb-N'80 is displayed in Fig. 2. The 554-cm-1 line, which is shifted to 545 cm-' by '5NO isotope (38), is practically insensitive (within ±1 cm-') to the NO -) N180 substitution. Fig. 3 presents the low-frequency (200-700 cm-) resonance

Frequency (cm 1)

FIG. 2. Resonance Raman spectrum (200-650 cm-') of ferrous Mb-N80. Conditions same as in Fig. 1 except for the delay, 10,000; concentration, 60 ,uM in 0.05 M Tris-HCl, pH 7.2.

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Chemistry: Benko and Yu

200

300 400 500 600 Frequency (cm-1)

FIG. 3. NO isotope effects on resonance Raman spectra (200-650 cm ') of ferric HRP'NO. Conditions same as in Fig.A1 except for the delay, 10,000.

Raman spectra of ferric HRP-NO with various isotopes.. The two isotope-sensitive lines were detected at 604 and 574 cm-. Upon NO -) 15NO substitution, the 604-cm-1 line shifts to lower wavenumbers ( 598 cm-1), becoming closer to the more intense porphyrin ring mode and resulting in the asymmetric line at 592 cm-1. The broadening of this line on the high-frequency side diminishes considerably from 15NO to N180 because of a further shift, which. causes a more complete overlap of the two lines. However, the 574-cm-1 line shifts to -571 cm-' (15NO), which then increases to 574 cm-1. It should be noted that there is a reproducible broadening on the low-frequency side of the 571-cm-' line (middle curve, Fig. 3), which suggests the existence of two overlapping lines at -574 cm-'. The isotopesensitive compronent shifts to =564 cm-1 (15NO) and then to 574 cm-' (N1 0). Interestingly, the 574-cm-1 line exhibits no isotope shift in going from NO to N180. In Fig. 4 we present six spectra (1,400-1,700 cm-') taken at different time intervals after the addition of NO to met-Mb. The most striking changes occur at 1,512 and 1,647 cm-l, which shift to 1,502 and 1,636 cm-1, respectively, upon autoreduction. However, there is no frequency shift at 1,375 cm-1 between ferric Mb-NO and ferrous Mb-NO. The absence of aRaman line at -1,362 cm-1 in the intermediate spectra-indicates

1350. 1400 1500 -1 60 Frequency. (cm-1)

FIG. 4. Time-resolved resonance Raman spectra (1,300-1,700 cmn') in the conversion of ferric Mb NO to ferrous Mb-NO. Conditions same as in Fig. 1.

Proc. Natl. Acad. Sci. USA 80 (1983)

) 1500. 1600 '1700 Frequency (cm-1)

FIG. 5. Time-resolved resonance Raman spectra (1,400-1,700 cmnf) in the conversion of ferric HbA-NO to ferrous HbA-NO [in the presence of inositol hexaphosphate (IHP)I. Conditions same as in Fig. 1. Concentration, 85 PM (heme basis), 5 mM inosit6l hexaphosphate, 0.05 M phosphate buffer, pH 6.0.

that there is no appreciable concentration of a steady-state Fe(II).NO+ intermediate (14, 15); although kinetically such a transient species may be present at low concentration. Similar results for HbA-NO are shown in Fig.: 5. -In the. low-frequency region, there is an isotope-sensitive line, at 594 cm-1 in ferric HbA-NO (plus inositol hexaphosphate) (not shown) and at 551 cm 1 in ferrous HbA-NO as reported previously (38}. The addition of inositol hexaphosphate decreases the rate of autoreduction. In contrast, the ferric HRP-NO complex is quite stable (19); only two spectra (i.e., ferric and ferrous) are compared in Fig. 6. The two porphyrin ring modes at 1,514 and 1,644 cm-' in ferric- HRPNO are very similar to those at 1,512 and 1,647 cm 1 (ferric Mb-NO), or at 1,507 and 1,643 cm-' (in ferric HbA.NO). Unlike the conversion of low-spin Fe(III) to low-spin Fe(II) in the cyanide complex of HRP, in which there is a frequency shift from 1,375 to 1,362 cm-1 (53), the addition of one electron to the ferric Mb-NO complex does not alter the frequency at 1,378 cm-' (within ± 1 cm-'). The difference in charge between the two oxidation states is absorbed by both the NO ligand and the proximal histidine, without affecting the 9r* electron density.of the porphyrin macrocycle (1).-

10 1400 1500 Frequency (cm- 1) FIG. 6. Comparison of resonance Raman spectra of ferric and ferrous HRP-NO (1,300-1,700 cm'). Conditions: excitation A; 413.1 nm; power, 7 mW; delay, 10,000; concentration, 20 pM HIRP, 0.05 M Tris-HCl, pH 8.2.

Chemistry:

Benko and Yu DISCUSSION

Bonding Interactions Between Fe(III) and NO; Assignment of Fe(III)-NO Stretching and Fe(Il)-N-o Bending Vibrations. The-binding of nitric oxide to the ferric forms of Mb, Hb, and HRP results in the formation of diamagnetic complexes (14, 15, 19). The original ferric hemoproteins can be recoveredFfrom the-ferric NO complexes when the NO is removed by degassing, indicating the reversible nature of the interactions between Fe(III) and NO (14,415, 19, 20). Interest in the ferric-NO complexes stems from the fact that the [Fe(III) + NO] and [Fe(II) + CO] systems are isoelectronic, having a total of six electrons associated with the metal d and ligand A* orbitals; a linear Fe(III)-NO linkage is expected (20, 40) in the absence of a distal steric effect. Recent resonance Raman studies of HbA-CO and Mb-CO (4) have led to the definite identification of the Fe(II)-CO stretching and Fe(II)C--O bending vibrations; it should be of great interest to see if the Fe(III)-NO stretching and Fe(III)-N-O bending modes can be resonance-enhanced through Soret excitation. A -comparison of these vibrational frequencies between the two systems should provide new insights into the nature of these iron-axial-ligand bonds in hemoproteins. Indeed, we have detected two isotope-sensitive lines at 595 and 573 cm-' in the resonance Raman spectrum of ferric MbhNO excited at 406.7 nm (Fig. 1). Analogous to the vibrational assignments for carbonmonoxy hemes (4, 54), we assign the 595cm-' line to theFe(III)-NO stretching mode and the 573 cm-' line to the Fe(III)-N--)O bending vibration. Recently, Walters and Spiro (39), on the basis of one isotope shift, assigned two similar frequencies at 596 and 573 cm-1 to the Fe(II)-NO stretching and Fe(II)-N-O bending vibrations, respectively, in the so-called "penta-coordinate ferrous MbNO complex at pH 5.8". However, EPR studies (29) showed no conversion of hexa- to pentacoordinated ferrous Mb-NO at pH 5.8. Mackin et al. (55) found that Walters and Spiro's sample was a mixture Nature of

of ferric and ferrous Mb-NO and that there was no Soret-excited resonance Raman enhancement of the 596- and 573-cm-1 lines in a pentacoordinated ferrous Mb-NO prepared by interactions with sodium dodecyl sulfate. Therefore, we conclude that their isotope-sensitive lines at 596 and 573 cm-1 are due to the same ferric Mb-NO complex as in this study. In addition, we note that the pentacoordinated Fe(II)-NO stretch at 592 cm'1 in ferrous Hb-NO (with inositol hexaphosphate) reported by Stong et al. (36) is very similar to the 594-cm-1 line we have detected in ferric Hb-NO (with inositol hexaphosphate). At present, -we do not have evidence that their assignment may be invalid, especially because their excitation wavelength at 454.5 nm is quite different from the 406.7 nm used here. In the case of ferric HRP NO (Fig. 3) we assign the 604-cm-1 line to the Fe(III)-NO stretching and the 574-cm-1 line to the Fe(III)-N--O bending mode. Although the Fe(III)-NO stretch is 9 cm-' higher in ferric HRP-NO than in ferric MbWNO, the Fe(III)-N--O bending frequencies are nearly the same in these two complexes. More interesting is the comparison between ferric Mb-NO and Mb-CO, in which the Fe(II)-C-O bending mode at =577 cm-' is similar to the Fe(III)-N--O bending mode at 573 cm-1; however, the stretching frequencies of the Fe(II)-CO and Fe(III)-NO bonds differ by 83 cm-! Assuming that both Fe(III)-NO and Fe(ll)-CO linkages are linear and the small distortion by the protein is similar, the stretching frequencies at =600 cm-1 for the Fe(III)-NO bond and at =500 cm-' for the Fe(II)-CO bond may be employed to estimate the relative force constants. Based on a diatomic model considering CO or NO as a single mass, the force constant for the Fe(III)-NO bond is 1.5 times greater than that

Proc. Natl. Acad. Sci. USA 80 (1983)

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for the Fe(II)-CO bond. It is generally observed that the greater the force constant, the shorter the bond length and the greater the bond strength (56). Thus, we believe that the Fe(III)-NO bond is shorter (hence stronger) than the Fe(II)-CO bond. The origin of the greater bond strength for the Fe(III)-NO bond compared to the Fe(II)-CO bond may reside in the participation in ir bonding by the unpaired electron in the ir* (NO) orbital. Upon binding, this electron coupled to the unpaired d4 electron of the heme iron (i.e., antiferromagnetically coupled) forms a strong 7r bond between the Fe(III) and -NO ligand, in ,addition to the o bond. The enhancement of the Fe(III)-N-O bending mode in the ferric-NO complexes of Mb and HRP is of considerable interest in view of a recent study on sterically hindered carbonmonoxy hemes by Yu et al. (54), who demonstrated that the Fe(II)-CO distortion-by steric interactions induces the resonance Raman enhancement of the Fe(II)-C-O bending mode. By analogy, we suggest that the observation of the Fe(III)-N-O bending mode at 573 cm-1 is indicative of the Fe(III)-N-O distortion in nitrosyl ferric hemoproteins. Assignment of Fe(l)-N-O Bending Vibration in Ferrous Mb NO. The assignment of the Fe(II)-NO stretching mode at =550 cm-' in the resonance Raman spectrum of ferrous HbA-NO was proposed by Chottard and Mansuy (8), and supported by Stong et al. (36) and Tsubaki and Yu (38). It has been generally accepted without question (1-3, 32, 36-39). The assignment was based on one isotope shift (i.e., NO -- 15NO substitution), whichdid not distinguish the Fe-NO stretching from the Fe-N--O bending mode. We employed here a terminally labeled isotope (N`8O) and were surprised to find that the 554cm1 line in the ferrous Mb'NO spectrum is insensitive to such a substitution. In the order of increasing mass (NO -- 15NO -_ N180) the frequency shifts (554 -- 545 -- 554 cm-') exhibit a zigzag pattern, suggestive of an Fe(II)-N--O bending mode. The only condition in which the Fe(II)-NO stretching frequency would be insensitive to the isotope substitution at the terminal oxygen is that the Fe- N-O angle (6) is 900, which is highly unlikely in view of the reported 0 of 1420 in the model complex Fe(H) (tetraphenylporphyrin) (N-methylimidazole) (NO) (23), and 1530 in Mb-NO (30). When 0 is smaller than 1500 there is a considerable mixing between the stretching and bending modes. However, normal coordinate calculations (unpublished results) based on the model (imidazole)-Fe N--O indicated that it is still possible to assign a normal mode as either predominantly stretching or predominantly bending on the basis of potential energy distributions. The fact that only one mode is preferentially resonance enhanced in ferrous Mb-NO suggests that the mixing between stretching and bending may not be too extensive. § Therefore, on the basis of the observed zigzag isotope shifts in the order (NO -- 15NO -* N180) we reassign the 554 cm-1 line in ferrous Mb-NO as the Fe(Il)-N--O bending vibration rather than the Fe(II)-NO stretching mode. Assignment of Fe(II)-O--O Bending Vibration in Oxy Hemoproteins. The Fe(II)-02 stretching vibration at 567 cm-l in oxy-Hb was identified by Brunner (9) via its isotope shift to 540 cm' upon 1602 ->1802 substitution. Subsequently, the same vibration at similarfrequencies has been reported for oxy-Mb (32), oxyleghemoglobin (42), and the oxy adduct of synthetic "picket fence" porphyrin (45). Perhaps it is the most widely accepted assignment in resonance Raman spectroscopy of hemoproteins (1-4, 6-9, 32, 37, 39, 41-46). Because of its impor§ The simultaneous observation of both stretching and bending in Mb CO

and ferric Mb NO does not necessarily mean that the mixing is more extensive. In fact, we expect a lesser degree of mixing there because of the more linear ligand geometries.

7:Proc. Nati. Acad. Sci. USA 80 (1983) 7046 Chemistry: Benko and Yu tance as .a monitor of the Fe(II)02 bond strength, the assignment should be reassessed in the light of the present studies on nitrosyl hemoproteins. In 1979, Duff et al. (46) studied -the Fe(Ii-02 stretching frequency in oxy-HbA containing isotopically unsymmetric dioxygen (60180), in an attempt to distinguish an end-on geometry from a side-on one. They observed two Raman peaks at 567 and 540 cm1 in Hb 160180 as compared. with 567 cm1 in HbV602 and 540 cm-' in Hbl1802. The two peaks were interpreted as due to the following two species: Fe(II)l-60150 (567 cm-) and Fe(II)-`80 60 (540 cm' ) in favor of an endon geometry. However, they were unable to explain-why the Fe(II)-02 stretching frequency is insensitive to the terminal isotope label; i.e., why v(Fe-'60180) is the same as v(Fe-'160160) and k(Fe-'80160) is the same as v (Fe-180180). In particular, their simple valence force field calculation (46) revealed that there should be an 8-cm-' isotope shift in going from Fe(II)-1602 to Fe(II) 160180, assuming the Fe(II)0-0 angle of 1350, which is even smaller than the 1560 value found in oxy-HbA by a recent x-ray crystallographic study (57).

These discrepancies between calculations and experimental data may be resolved if the 567-cm-1 line is assigned to the Fe(II)-0-0 bending mode. The evidence is clear: in the order (1602 160180 the frequency shifts 180160 _1802) (567 -* 540 -* 567 -) 540 cm-l) display a zigzag patternm analogous to those found for Fe(II)-N--0 bending, Fe(II)---0 bending, and Fe(III)-N-0 bending vibrations. As far as the results of Duff et al. (46) are valid, we may conclude that the 567-cm'- line in oxy-HbA is the Fe(II)-0--0 bending mode. Further experiments, using 160'80-labeled model compounds such as oxy-Fe(II) picket-fence porphyrins in which this isotope-sensitive line is stronger and sharper (58) than in oxy-HbA/ oxy-Mb, will be helpful in definitely assigning this line as bending. ¶ Unlike '5NO and N180, 180160 and 16018O have the same mass. One would expect a small increase in the Fe-02 stretching frequency in going from Fe-180'60 to Fe 160180 because the Fe O-O angle is 156°, less than 1800 (57). We thank Helen C. Mackin and Ellen A. Kerr for excellent technical assistance. Comments and suggestions by Miss Helen C. Mackin are greatly appreciated. This research was supported by Grant GM 18894 from the National Institutes of Health. 1. Rousseau, D. L. & Ondrias, M. R. (1983) Annu. Rev. Biophys.

Bioeng. 12, 357-380. 2. Spiro, T. G. (1983) in Iron Porphyrins, -eds. Lever, A. B. P. & Gray, H. B. (Addison-Wesley, Reading, MA), Part 2, pp. 89-152. 3. Asher, S. A. (1982) Methods Enzymol. 76, 371-413. 4. Tsubaki, M., Srivastava, R. B. & Yu, N.-T. (1982) Biochemistry 21, 1132-1140. 5. Champion, P. M., Stallard, B. R., Wagner, G. C. & Gunsalus, I. C. (1982) J. Am. Chem. Soc. 104, 5469-5472. 6. Nagai, K., Kitagawa, T. & Morimoto, H. (1980)J. Mol. Biol. 136, 271-289. 7. Hori, H. & Kitagawa, T. (1980) J. Am. Chem. Soc. 102, 3608-3613. 8. Chottard, G. & Mansuy, D. (1977) Biochem. Biophys. Res. Commun. 77, 1333-1338. 9. Brunner, H. (1974) Naturwissenschaften 61, 129-130. 10. Tsubaki, M., Srivastava, R. B. & Yu, N.-T. (1981) Biochemistry 20, 946-952. 11. Yu, N.-T. & Tsubaki, M. (1980) Biochemistry 19, 4647-4653. 12. Keilin, D. & Mann, T. (1937) Proc. Roy. Soc. London Ser. B. 122, 119-133. 13. Keilin, D. & Hartree, E. F. (1937) Nature (London) 139, 548-563. 14. Ehrenberg, A. & Szczepkowski, T. W. (1960) Acta Chem. Scand. 14, 1684-1692. 15. Butt, W. D. & Keilin, D. (1962) Proc. Roy. Soc. London Ser. B 156, 429-458. 16. Kon, H. (1960) Biochem. Biophys. Res. Commun. 35, 423-427.

17. Wittenberg, J. B., Noble, R. W., Wittenberg, B. A., Antonini, E., Brunori, M. & Wyman, J. (1967)J. Bio,. Chem. 242, 626-634. 18. Chien, J. C. W. (1969)J. Am. Chem. Soc. 91, 2166-2168. 19. Yonetani, T., Yamamoto, H., Erman, J. E., Leigh, J. S., Jr., & Reed, G. H. (1972) J. Biol. Chem. 247, 2447-2455.

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