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

Resonance Raman studies of nitric oxide binding to ferric andferrous hemoproteins: Detection of Fe(III)-NO stretching,Fe(III)-N-O beding, and Fe(II)-N-Obending vibrations

(myoglobin/hemoglobin/horseradish peroxidase/ligand vibrations)

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

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

ABSTRACT The nature of bonding interactions betweenFe(EI) and NO in the ferric nitrosyl complexes of myoglobin (Mb),hemoglobin A (HbA), and horseradish peroxidase (HRP) is inves-tigated by Soret-excited resonance Raman spectroscopy. On thebasis of 5NO and N'80 isotope shifts, we clearly identified theFe(IE)-NO bond stretching frequencies at 595 cm-1 (ferricMb'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 verysimilar to the Fe(l)-C-O bending modes at 578 cm1 in Mb'COand HbA-CO. However, the Fe(III)-NO and Fe(II)-COstretching frequencies differ by -90 cm-f, indicating a muchstronger 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 ab-sence of distal steric effect). The unusually strong Fe(IH)-NObond may be attributed to the ir bondin involving the unpairedelectron in the ir*(NO) orbital. The N1 0 isotope shift data in-dicate that the widely accepted assignment of the Fe(II)-NOstretching vibration at "554 cm-' in ferrous nitrosyl Mb/HbA isincorrect; instead, we assign it to the Fe(il)-N-O bending mode.The validity of the assignment of Fe(i)-02 stretch at 567 cm-in oxy-HbA by Brunner [Brunner, H. (1974) Naturwissenschaften61, 129-130] is now in doubt. Literature data are presented tosuggest that it is the Fe(Il)--O-O bending vibration.

Resonance Raman spectroscopy is an ideal tool for directlymonitoring the iron-axial ligand bond strength in hemoproteinsand synthetic model hemes (refs. 1-3 and references citedtherein). The information it provides is important in under-standing the mechanisms of protein control of heme reactivity.At present, there are several stretching vibrations of iron-axialligand bonds that have been unambiguously identified. For ex-ample, the Fe-C bond in (carbonmonoxy) hemoglobin (Hb)and myoglobin (Mb) (4), Fe-S bond in cytochrome P450 (cam-phor) (5), Fe-Ne (proximal histidine) bond in deoxy-Hb/Mb(6, 7), and Fe-CN bond in cyanometerythrocruorin (unpub-lished results). The assignments of Raman lines characteristicof these vibrations were made through at least two differentisotope shifts. However, there are important assignments suchas the Fe(II)-NO stretch in Hb-NO (8) and the Fe(II)-02stretch in Hb-02 (9) that were based on a single isotope shift.Such an experimental approach does not enable one to distin-guish between iron-ligand stretching and bending vibrations.On the basis of a tacit assumption that the iron-ligand stretch-ing vibration should be more readily enhanced than the bend-

ing mode, investigators often assigned the only isotope-sensi-tive line in the 100- to 700-cm-1 region as the iron-ligandstretching mode. However, it has been demonstrated (10, 11)that, in azide complexes of metmyoglobin, methemoglobin, andmanganese-substituted myoglobin, only ligand bending modeswere enhanced when the metal-ligand stretching modes werenot detectable.

Nitric oxide binds to both ferric and ferrous hemoproteins(12-20). Although ferrous nitrosyl complexes have been exten-sively studied by x-ray diffraction (21-24), infrared (24, 25), vis-ible absorption (26-28), EPR (19, 20, 22, 29, 30), extended x-ray absorption fine structure (EXAFS) (31), and resonance Ra-man spectroscopy (8, 32-39), very little is known regarding thebonding interactions between Fe(III) and NO in hemoproteinsand heme model compounds, presumably because of the in-trinsic tendency towards spontaneous autoreduction (12-20),the diamagnetic property (EPR silent) (14, 15), and the highquantum yield of ligand photodissociation (40) have hinderedmeaningful resonance Raman studies.

In this paper, we demonstrate the feasibility of obtaining high-quality resonance Raman spectra of nitrosyl complexes of Mb,hemoglobin A (HbA), and horseradish peroxidase (HRP) in bothferric and ferrous forms. The Fe(III)-NO stretch has beenclearly identified (by using '5NO and N180 isotopes) at -600cml, which is much higher than the Fe(II-CO stretch at =500cm-' (4), the Fe(III)-S stretch at =350 cm-' (5), and theFe(III)-CN- stretch at 454 cm-' (unpublished results). TheFe(III)-N--O bending vibration is assigned to the =573-cm 1line on the basis of its "zigzag" pattern. We were surprised tofind that a similar zigzag pattern exists with the ==554-cm-1 linein ferrous nitrosyl Hb/Mb; this line was previously assigned tothe Fe(II)-NO stretch by Chottard and Mansuy (8). Here, weassign it to the Fe(II)-N--O bending mode. The generallyaccepted assignment of the Fe(II)-02 stretch at =570 cm-1in oxy-Hb/Mb (1-4, 7-9, 32, 37, 39, 41-46) is reassessed byusing the Raman data from oxy-Hb with 16018O isotope (46); itis suggested as the Fe(II)-O--O bending mode.

MATERIALS AND METHODSSperm whale Mb (Sigma) was purified in the ferric form by theprocedure described previously (11). Human hemoglobin Ao(HbA) was prepared in the oxy form from whole blood as de-scribed 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.

7042

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Proc. Natl. Acad. Sci. USA 80 (1983) 7043

ferric form by oxidation with potassium ferricyanide, which wassubsequently removed by extensive dialysis against 0.05 M so-dium phosphate buffer at pH 6.0. HRP from Worthington (A4M/A275 = 3.16; activity = 1,058 units/mg) was purified on Seph-adex A-50 and CM-cellulose column according to Paul and Stig-brand (48); only C type HPR was used in this study. Heme con-centrations were determined spectrophotometrically, with thefollowing extinction coefficients: 11.0 mM'1 cm-' at 540 nm(ferric MbCN- and ferric HbCN-) (49) and 100 mM-1 cm-1at 403 nm (resting HRP) (48). For the time-dependence studyof the autoreduction of ferric Mb-NO or ferric HbA-NO, thesample solution (in ferric form) was diluted to the desired con-centration (60-90 ,uM) with appropriate buffer and then trans-ferred to a Raman cell fitted with a rubber septum. The oxygenin the solution was removed by repeated evacuation and flush-ing with pure nitrogen gas (Matheson, CP grade). After the fi-nal evacuation, nitric oxide (Matheson, CP grade) washed withconcentrated NaOH solution (Q0. 1 M) was introduced at apressure of %z1 atmosphere (101 kPa) or slightly less. To studyferric Mb-NO with different isotopes (14N160, 15N'60, and14N'80), Raman spectra were recorded within less than 7 minafter the mixing of ferric Mb solution with NO gas. After =8hr of standing at room temperature, the sample of ferric Mb-NO(or ferric HbA-NO) was spontaneously converted to ferrousMb-NO, without addition of sodium dithionite (12-15). Forcontrol experiments the ferric form of Mb (or HbA) was an-aerobically reduced in the Raman cell by injecting a slight ex-cess of sodium dithionite solution (buffered and degassed) be-fore nitric oxide was introduced. The two methods producedidentical resonance Raman spectra of ferrous Mb-NO (or HbA-NO).The samples of nitric oxide composed of different isotopes

were obtained from the following sources: 15NO (Stohler Iso-tope Chemicals, Waltham, MA; 99% enrichment in '5N) andN'80 (custom-synthesized by Prochem US Services, Summit,NJ; 90% enrichment in 180).

Resonance Raman spectra were recorded with a highly sen-sitive multichannel system (S0) consisting of a modified Spex1402 0.85-m Czerny-Turner double monochromator (two 600grooves per mm gratings in additive dispersion), a dry ice-cooled(-620C) intensified vidicon detector (Princeton Applied Re-search model 1254), a PAR 1216 detector controller, a Tektronix604 monitor, and a PAR model OMA 2 microprocessor-basedconsole. Light at the excitation wavelengths 406-.7 and 413.1nm was provided by a Spectra-Physics model 171 Kr' laser.The ligand photodissociation, especially the ferric Mb-NO hav-ing a quantum yield of -1 (40), was minimized by spinning theRaman cell (=2,000 rpm) during laser irradiation. The slit widthused was 100 jAm and the slit height was 2 mm. Spectra werecalibrated by using fenchone as a standard compound (50). Ad-ditional standards used for the high-frequency region were tol-uene (1379, 1385, and 1604 cm-') or benzene (1585 and 1606cm'1). The Raman spectra presented here have not been com-puter-smoothed. Reported wavenumbers are accurate within±1 cm-1 for sharp lines and ± 2 cm-1 for broad lines.

RESULTSResonance Raman spectra (200-650 cm-') of ferric Mb-NO withvarious isotopes are presented in Fig. 1. Spectra were obtainedwithin 7 min after the addition of NO; there is no significantamount of ferrous species present, as judged by the absence ofa distinct line at 357 cm-'. There were virtually no changes inthe spectrum when the laser power was increased from 5 to 30mW, nor were any changes observed when the pH was variedbetween 8.4 and 5.8. There are two isotope-sensitive lines at

200 300 400 500 600Frequency (cm-1)

FIG. 1. NO isotope effects on resonance Raman spectra (200-650cmn1) of ferric Mb-NO. Conditions: excitation A, 406.7 nm; power, 15mW; delayed cycles, 1,000 (30.3 sec); slit width, 100 pam; slit height, 0.2cm; 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) and587 cm-' (N'80) as the NO mass increases by 1 and 2 daltons,respectively. In contrast, the 573-cm-1 line decreases to 562cm-' (5NO), which then increases to 569 cm-1 (N180). On thebasis of the observed shift from NO to 15NO, one would expectthe frequency to decrease again from N'80 to 15N180. The re-maining features in the 200- to 650-cm-1 region are typical offerric Mb derivatives such as aquo-, fluoro-, azido-, and hy-droxyl-met-Mb (unpublished results). The 411-cm-1 line maybe assigned to the vinyl group bending mode on the basis ofrecent 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 theferrous Mb-NO species. The resulting spectra with NO and "5NOare identical to those reported recently from this laboratory (38).Contrary to the work of Walters and Spiro (39), we found nopH dependence of ferrous Mb-NO spectrum between 8.4 and5.8.

The resonance Raman spectrum of ferrous Mb-N'80 is dis-played in Fig. 2. The 554-cm-1 line, which is shifted to 545cm-' 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 ferrousMb-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.

Chemistry: Benko and Yu

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

200 300 400 500 600Frequency (cm-1)

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

Raman spectra of ferric HRP-NO with various isotopes.. Thetwo isotope-sensitive lines were detected at 604 and 574 cm-.Upon NO -) 15NO substitution, the 604-cm-1 line shifts to lowerwavenumbers ( 598 cm-1), becoming closer to the more in-tense porphyrin ring mode and resulting in the asymmetric lineat 592 cm-1. The broadening of this line on the high-frequencyside diminishes considerably from 15NO to N180 because of afurther shift, which. causes a more complete overlap of the twolines. However, the 574-cm-1 line shifts to -571 cm-' (15NO),which then increases to 574 cm-1. It should be noted that thereis a reproducible broadening on the low-frequency side of the571-cm-' line (middle curve, Fig. 3), which suggests the ex-istence of two overlapping lines at -574 cm-'. The isotope-sensitive compronent shifts to =564 cm-1 (15NO) and then to574 cm-' (N1 0). Interestingly, the 574-cm-1 line exhibits noisotope shift in going from NO to N180.

In Fig. 4 we present six spectra (1,400-1,700 cm-') taken atdifferent time intervals after the addition of NO to met-Mb.The most striking changes occur at 1,512 and 1,647 cm-l, whichshift to 1,502 and 1,636 cm-1, respectively, upon autoreduc-tion. However, there is no frequency shift at 1,375 cm-1 be-tween ferric Mb-NO and ferrous Mb-NO. The absence of aRa-man line at -1,362 cm-1 in the intermediate spectra-indicates

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

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

that there is no appreciable concentration of a steady-stateFe(II).NO+ intermediate (14, 15); although kinetically such atransient species may be present at low concentration. Similarresults for HbA-NO are shown in Fig.: 5. -In the. low-frequencyregion, there is an isotope-sensitive line, at 594 cm-1 in ferricHbA-NO (plus inositol hexaphosphate) (not shown) and at 551cm 1 in ferrous HbA-NO as reported previously (38}. The ad-dition of inositol hexaphosphate decreases the rate of autore-duction. In contrast, the ferric HRP-NO complex is quite stable(19); only two spectra (i.e., ferric and ferrous) are compared inFig. 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 1,647cm 1 (ferric Mb-NO), or at 1,507 and 1,643 cm-' (in ferricHbA.NO). Unlike the conversion of low-spin Fe(III) to low-spinFe(II) in the cyanide complex of HRP, in which there is a fre-quency shift from 1,375 to 1,362 cm-1 (53), the addition of oneelectron to the ferric Mb-NO complex does not alter the fre-quency at 1,378 cm-' (within ± 1 cm-'). The difference in chargebetween the two oxidation states is absorbed by both the NOligand and the proximal histidine, without affecting the 9r*electron density.of the porphyrin macrocycle (1).-

1350. 1400 1500 -160Frequency. (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 sameas in Fig. 1.

10 1400 1500Frequency (cm- 1)

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

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Proc. Natl. Acad. Sci. USA 80 (1983) 7045

DISCUSSIONNature of Bonding Interactions Between Fe(III) and NO;

Assignment of Fe(III)-NO Stretching and Fe(Il)-N-oBending Vibrations. The-binding of nitric oxide to the ferricforms of Mb, Hb, and HRP results in the formation of dia-magnetic complexes (14, 15, 19). The original ferric hemopro-teins can be recoveredFfrom the-ferric NO complexes when theNO is removed by degassing, indicating the reversible natureof 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 andligand A* orbitals; a linear Fe(III)-NO linkage is expected (20,40) in the absence of a distal steric effect. Recent resonanceRaman studies of HbA-CO and Mb-CO (4) have led to the def-inite identification of the Fe(II)-CO stretching and Fe(II)-C--O bending vibrations; it should be of great interest to seeif the Fe(III)-NO stretching and Fe(III)-N-O bendingmodes can be resonance-enhanced through Soret excitation. A-comparison of these vibrational frequencies between the twosystems should provide new insights into the nature of theseiron-axial-ligand bonds in hemoproteins.

Indeed, we have detected two isotope-sensitive lines at 595and 573 cm-' in the resonance Raman spectrum of ferric MbhNOexcited at 406.7 nm (Fig. 1). Analogous to the vibrational as-signments for carbonmonoxy hemes (4, 54), we assign the 595-cm-' line to theFe(III)-NO stretching mode and the 573 cm-'line to the Fe(III)-N--)O bending vibration. Recently, Wal-ters and Spiro (39), on the basis of one isotope shift, assignedtwo similar frequencies at 596 and 573 cm-1 to the Fe(II)-NOstretching and Fe(II)-N-O bending vibrations, respectively,in the so-called "penta-coordinate ferrous MbNO complex atpH 5.8". However, EPR studies (29) showed no conversion ofhexa- to pentacoordinated ferrous Mb-NO at pH 5.8. Mackinet al. (55) found that Walters and Spiro's sample was a mixtureof ferric and ferrous Mb-NO and that there was no Soret-ex-cited resonance Raman enhancement of the 596- and 573-cm-1lines in a pentacoordinated ferrous Mb-NO prepared by inter-actions with sodium dodecyl sulfate. Therefore, we concludethat their isotope-sensitive lines at 596 and 573 cm-1 are dueto the same ferric Mb-NO complex as in this study. In addition,we note that the pentacoordinated Fe(II)-NO stretch at 592cm'1 in ferrous Hb-NO (with inositol hexaphosphate) reportedby Stong et al. (36) is very similar to the 594-cm-1 line we havedetected in ferric Hb-NO (with inositol hexaphosphate). Atpresent, -we do not have evidence that their assignment maybe invalid, especially because their excitation wavelength at454.5 nm is quite different from the 406.7 nm used here. Inthe case of ferric HRP NO (Fig. 3) we assign the 604-cm-1 lineto the Fe(III)-NO stretching and the 574-cm-1 line to theFe(III)-N--O bending mode.

Although the Fe(III)-NO stretch is 9 cm-' higher in ferricHRP-NO than in ferric MbWNO, the Fe(III)-N--O bendingfrequencies are nearly the same in these two complexes. Moreinteresting is the comparison between ferric Mb-NO and Mb-CO,in which the Fe(II)-C-O bending mode at =577 cm-' is sim-ilar to the Fe(III)-N--O bending mode at 573 cm-1; however,the stretching frequencies of the Fe(II)-CO and Fe(III)-NObonds differ by 83 cm-!

Assuming that both Fe(III)-NO and Fe(ll)-CO linkagesare linear and the small distortion by the protein is similar, thestretching frequencies at =600 cm-1 for the Fe(III)-NO bondand at =500 cm-' for the Fe(II)-CO bond may be employedto estimate the relative force constants. Based on a diatomicmodel considering CO or NO as a single mass, the force con-stant for the Fe(III)-NO bond is 1.5 times greater than that

for the Fe(II)-CO bond. It is generally observed that the greaterthe force constant, the shorter the bond length and the greaterthe bond strength (56). Thus, we believe that the Fe(III)-NObond is shorter (hence stronger) than the Fe(II)-CO bond.The origin of the greater bond strength for the Fe(III)-NObond compared to the Fe(II)-CO bond may reside in the par-ticipation in ir bonding by the unpaired electron in the ir* (NO)orbital. Upon binding, this electron coupled to the unpaired d4electron 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 inthe ferric-NO complexes of Mb and HRP is of considerable in-terest in view of a recent study on sterically hindered carbon-monoxy hemes by Yu et al. (54), who demonstrated that theFe(II)-CO distortion-by steric interactions induces the reso-nance Raman enhancement of the Fe(II)-C-O bendingmode. By analogy, we suggest that the observation of theFe(III)-N-O bending mode at 573 cm-1 is indicative ofthe Fe(III)-N-O distortion in nitrosyl ferric hemoproteins.

Assignment of Fe(l)-N-O Bending Vibration in FerrousMb NO. The assignment of the Fe(II)-NO stretching modeat =550 cm-' in the resonance Raman spectrum of ferrousHbA-NO was proposed by Chottard and Mansuy (8), and sup-ported by Stong et al. (36) and Tsubaki and Yu (38). It has beengenerally accepted without question (1-3, 32, 36-39). The as-signment was based on one isotope shift (i.e., NO -- 15NO sub-stitution), whichdid not distinguish the Fe-NO stretching fromthe Fe-N--O bending mode. We employed here a terminallylabeled isotope (N`8O) and were surprised to find that the 554-cm1 line in the ferrous Mb'NO spectrum is insensitive to sucha substitution. In the order of increasing mass (NO -- 15NO -_N180) the frequency shifts (554 -- 545 -- 554 cm-') exhibit azigzag pattern, suggestive of an Fe(II)-N--O bending mode.The only condition in which the Fe(II)-NO stretching fre-quency would be insensitive to the isotope substitution at theterminal oxygen is that the Fe- N-O angle (6) is 900, whichis highly unlikely in view of the reported 0 of 1420 in the modelcomplex Fe(H) (tetraphenylporphyrin) (N-methylimidazole) (NO)(23), and 1530 in Mb-NO (30). When 0 is smaller than 1500 thereis a considerable mixing between the stretching and bendingmodes. However, normal coordinate calculations (unpublishedresults) based on the model (imidazole)-Fe N--O indicatedthat it is still possible to assign a normal mode as either pre-dominantly stretching or predominantly bending on the basisof potential energy distributions. The fact that only one modeis preferentially resonance enhanced in ferrous Mb-NO sug-gests that the mixing between stretching and bending may notbe too extensive. § Therefore, on the basis of the observed zig-zag isotope shifts in the order (NO -- 15NO -* N180) we reas-sign the 554 cm-1 line in ferrous Mb-NO as the Fe(Il)-N--Obending vibration rather than the Fe(II)-NO stretching mode.

Assignment of Fe(II)-O--O Bending Vibration in OxyHemoproteins. The Fe(II)-02 stretching vibration at 567 cm-lin oxy-Hb was identified by Brunner (9) via its isotope shift to540 cm' upon 1602 ->1802 substitution. Subsequently, the samevibration 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 ac-cepted assignment in resonance Raman spectroscopy of hemo-proteins (1-4, 6-9, 32, 37, 39, 41-46). Because of its impor-

§The simultaneous observation of both stretching and bending in Mb COand ferric Mb NO does not necessarily mean that the mixing is moreextensive. In fact, we expect a lesser degree of mixing there becauseof the more linear ligand geometries.

Chemistry: Benko and Yu

7:Proc.Nati. Acad. Sci. USA 80 (1983)

tance as .a monitor of the Fe(II)02 bond strength, theassignment should bereassessed in the light of the present studieson nitrosyl hemoproteins.

In 1979, Duff et al. (46) studied -the Fe(Ii-02 stretchingfrequency in oxy-HbA containing isotopically unsymmetric di-oxygen (60180), in an attempt to distinguish an end-on ge-ometry from a side-on one. They observed two Raman peaksat 567 and 540 cm1 in Hb 160180 as compared. with 567 cm1in HbV602 and 540 cm-' in Hbl1802. The two peaks were in-terpreted as due to the following two species: Fe(II)l-60150(567 cm-) and Fe(II)-`80 60 (540 cm' ) in favor of an end-on geometry. However, they were unable to explain-why theFe(II)-02 stretching frequency is insensitive to the terminalisotope label; i.e., why v(Fe-'60180) is the same asv(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 goingfrom Fe(II)-1602 to Fe(II) 160180, assuming the Fe(II)-0-0 angle of 1350, which is even smaller than the 1560 valuefound in oxy-HbA by a recent x-ray crystallographic study (57).

These discrepancies between calculations and experimentaldata may be resolved if the 567-cm-1 line is assigned to theFe(II)-0-0 bending mode. The evidence is clear: in the or-der (1602 180160 160180_1802) the frequency shifts(567 -* 540 -* 567 -) 540 cm-l) display a zigzag patternm anal-ogous to those found for Fe(II)-N--0 bending, Fe(II)---0bending, and Fe(III)-N-0 bending vibrations. As far as theresults of Duff et al. (46) are valid, we may conclude that the567-cm'- line in oxy-HbA is the Fe(II)-0--0 bending mode.Further experiments, using 160'80-labeled model compoundssuch as oxy-Fe(II) picket-fence porphyrins in which this iso-tope-sensitive line is stronger and sharper (58) than in oxy-HbA/oxy-Mb, will be helpful in definitely assigning this line as bend-ing.

¶ Unlike '5NO and N180, 180160 and 16018O have the same mass. Onewould expect a small increase in the Fe-02 stretching frequency ingoing from Fe-180'60 to Fe 160180 because the Fe O-O angleis 156°, less than 1800 (57).

We thank Helen C. Mackin and Ellen A. Kerr for excellent technicalassistance. Comments and suggestions by Miss Helen C. Mackin are

greatly appreciated. This research was supported by Grant GM 18894from the National Institutes of Health.

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