6652
J. Phys. Chem. 1994, 98, 6652-6660
Vibrational Assignments of the FeCO Unit of CO-Bound Heme Proteins Revisited Observation of a New CO-Isotope-Sensitive Raman Band Assignable to the FeCO Bending Fundamental S. Hirota,? T. Ogura,+**K. Shinzawa-Itoh,o S. Yoshikawa,o M. Nag&* and T. Kitagawa'*tp* Graduate University for Advanced Studies and Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki, 444 Japan, Department of Life Science, Faculty of Science, Himeji Institute of Technology, 1479- I Kanaji, Kamigoricho, Akogun. Hyogo. 678- 12 Japan, and Biological Laboratory, Kanazawa University School of Allied Medical Professions, Kanazawa. Ishikawa, 920 Japan Received: January 3, 1994; In Final Form: April 28, 1994'
In order to find the FeCO bending (&eo) fundamental, resonance Raman spectra in the 6OCk200-cm-I region of C O adducts of hemoglobin (Hb), its isolated chains, myoglobin (Mb), cytochrome c oxidase (CcO), and cytochrome P-450 (P-450) were reexamined. A new CO-isotope-sensitive band was found around 365 cm-I for all but MbCO. For HbCO this band was located at 367,355,363, and 353 cm-I for I2Cl60,13C160,l2Cl80, and 13C180adducts, respectively, and thus exhibited a zigzag pattern against the increase of the total mass of CO, similar to the 575-cm-I band which has been assigned to the d ~ d fundamental o hitherto. Relative intensity of the -365-cm-l band to the 575-cm-l band exhibited almost no change upon lowering of temperature from 20 to -10 OC, but since temperature dependent frequency shifts were appreciable, the present difference spectra could not determine if the intensity change by 1 1% expected for a difference combination band of a hot 210-cm-1 mode was involved or not. The maximum intensity enhancement of the 365-cm-I band in the excitation profile within the Soret band occurred at longer wavelengths than that of the 575-cm-' band. The frequency differences between the CO-isotope-sensitive band around 575 cm-1 and one around 365 cm-I were 208 f 3,213 f 5 , and 204 f 3 cm-* for all C O isotopes of HbCO, CcO-CO, and P-450C0, respectively. Despite that the Fe-CO and C-0 stretching modes exhibited no deuteration shifts, the frequency of the 575-cm-l band was higher in D2O than in HzO, and this feature was more prominent with MbCO than HbCO. The 575-cm-I band of MbCO was remarkably strong upon excitation at 406.7 nm, and this is distinct from other proteins examined. These observations suggest that the -365-cm-l band arises from the d ~ e o fundamental and the 575-cm-l band is its combination with a porphyrin vibration or possibly a Fe-C deformation mode. Normal-coordinate calculations for the isolated FeCO unit reasonably reproduce the observed isotopic frequency shifts under these assignments.
Introduction The carbon monoxide (CO) adduct of heme has fundamental importance in studies of heme proteins, since it has been extensively used to probe the structural characteristics in their dioxygen binding site. The vibrational frequencies of the FeCO unit obtained by resonance Raman (RR) and infrared (IR) spectroscopies, which include the CO stretching (YCO) around 19401980 cm-1, Fs-CO streching ( ~ ~ 4around 0 ) 470-520 cm-l, and Fe-C-O bending ( 6 ~ e o )around 550-580 cm-I, have served as indicators of interactions between the bound CO and the surrounding residues in the heme pocket.' However, the conclusion drawn from the RR and IR studies of CO-heme proteins would lose its foundation if the vibrationalassignments mentioned above were incorrect. In fact, the assignment of 6 ~ e ohas not been in full agreement. This paper is going to propose a new assignment about the fundamental on the basis of careful reexaminationof the RR spectra of CO-isotope adducts of a few heme proteins. Caughey and co-workers2 first pointed out that the IR YCO band is a sensitive probe of the distal pocket structure in hemoglobin (Hb) and myoglobin (Mb). Several groups= have noted that the Y F ~ frequencies O obtained by RR spectroscopy have a negative linear correlation with the IR YCO frequenciesfor a variety of h e m e 4 0 adducts, and accordingly uco and U F ~ provide the same kinds of information. Currently four different Author to whom correspondence should be addressed.
t Graduate University for Advanced Studies,Okazaki National Research
Institutes. t Institute for Molecular Science, Okazaki National Research Institutes. 1 Himeji Institute of Technology. 1 Kanazawa University School of Allied Medical Professions. *Abstract published in Aduance ACS Abstracts. June 1, 1994.
0022-365419412098-6652$04.50/0
CO conformersof mammalian Mb's and Hb's in the ground state are designated Ai in which the subscript specifies YCO frequencies: 1965 (&), 1942 (AI), 1949 (Az), and 1932 (AS)cm-1. It is worthwhile to note that A3 is a minority species in solution but is a major component in crystal form at low temperatures.7a.8~ This means that detailed structures of the FeCO unit of heme proteins in solutionsshould be discussed on the basis of the analysis of spectroscopic data for solutions. The FeCO unit of a free porphyrin prefers a linear upright geometry in solutions?JOand for such a structure the 6~e0 fundamental, belonging to the E species in C , symmetry, should be IR active but RR inactive. In fact, the protein-freeh e m e 4 0 complex with an upright geometry gives no RR band between 500 and 600 cm-l while it yields the Y F ~ RR O band at 495 cm-1 in an organic solution.llJ2 According to X-ray13 and neutron14 diffraction studies of sperm whale Mb crystal, the FeCO unit adopts the bent conformation in the protein, and the earlier studies's inferred that the binding of CO to Mb is selectively inhibited by steric repulsion from the distal histidine (E7-His) that impedes upright binding. On the basis of group theoretical considerations, the 6 ~ e ofundamental is RR active for this geometry and, accordingly, the CO-isotope-sensitiveRaman bands of sperm whale MbCO at 577 and 512 cm-1 have been assigned toSFaand vF~0,respectively.l~ TheCO-hemes with a (CH& chain strap on the CO binding site which sterically prohibits the upright geometry gave the 6 ~ e RR o band at 570-580 cm-l (with the stronger intensity for the shorter strap) besides the Y F ~ RR O band at 50G510 cm-1 (higher frequency for shorter strap).16b Consequently, it seemed reasonable also for proteins that the FeCO unit distorted by steric hindrance gives rise to a higher frequency for Y F and~stronger RR intensity for 6 ~ e compared o with nondistorted ones, although there was a suggestion that the 2b97,8
O
0 1994 American Chemical Society
FeCO Bending Fundamental of Heme Proteins 570-580-cm-1 band was too high to assign it to the fundamental and was possibly an overtone of aF&0.l7 Recently the V F ~ RR O band was investigated with various E7-mutantsof human Mb generatedby site-directed mutagenesis, and it was concluded that the bulkiness of E7-residue, and thus its steric hindrance, is not the main determinant of the V F frequency.18 Similar conclusions are obtained for YCO of sperm whale Mb mutants19 and human Hb mutants.” On the other hand, a relation between the V F ~ frequency O and the Fe-C-O bond angle or the tilting angle of the Fe-C bond has been investigated theoretically.1.5a.6J6 In these treatments a set of potential constants, which can well reproduce the frequencies observed for an equilibrium state, is used without changes of their values to predict the frequencies expected for distorted forms. Unfortunately,one c a l c ~ l a t i o npredicts ~ ~ that the V F frequency ~ becomes lower as the Fe-C-0 bending angle becomes smaller but another6 predicts the opposite trend. This discrepancy arises from the different assignments of the 6 ~ d RR o band. The correct force constant of 6~e0, which depends on the experimental assignment of the RR band, is indispensable to evaluate the distortion energy of the FeCO unit.21 On the other hand, spectroscopic analysis of the CO adduct of heme proteins is becoming increasingly important. According to recent biochemical studies,22 the endogenously produced CO is likely to be involved in neurotransmission, blood pressure regulation, and hemin catabolism by binding to a heme protein, guanilate cyclase. For further advanced application of RR spectroscopy to structural analysis of heme proteins, correct assignment of the 8 ~ RR~ band 0 is necessary. Accordingly, in this study, we carefully searched a new CO-isotope-sensitive RR band in a lower frequency region for various heme proteins including Hb, its a- and &chains, Mb, cytochrome c oxidase (CcO), and cytochrome P-450 (P-450). Here we propose reassignment of the band around 575 cm-I to a combinationband and a new band around 365 cm-l to the 6 ~ a fundamental 0 band.
Experimental Procedures Raman scattering was excited at 406.7 nm by a Kr+ ion laser (Spectra Physics, Model 2016), at 441.6 nm by a He/Cd laser (Kinmon Electrics, Model CD1805B), at 442.5-435.0 nm by a dye laser (Spectra Physics, Model 375B) with stilbene-420 pumped by an Ar+ ion laser (Spectra Physics, Model 2045). and at 426.0-427.0 nm by the second harmonic of the output of the Ar+ ion laser-pumpedTi-sapphire laser (Spectra Physics, Model 3900) through KNb03 crystal (VIRGO OPTICS, and detected by a charge-coupled device (CCD) (Astromed, CCD 3200) or a photodiode array (PAR 1421HQ) attached to a single monochromator (Ritsu Oyo Kogaku, DG-1000). This CCD, which exhibited much less cross-talk between neighboringchannels compared with the diode array, was used to observe Raman bands with narrower bandwidths, while thediode array, which apparently exhibited higher sensitivity than CCD, was used to detect weak bands with better signal to noise (S/N) ratios. The slit width of 200 pm and slit height of 10 mm were used throughout the measurements, and the laser power at the sample point was adjusted to be 1-6 mW with a continuousND filter for the beam diameter of ca. 50 pm. RR spectra were measured at room temperature with a spinning cell (3000 rpm) to avoid photodissociation, except for the experiments for examining temperature dependences for which the spinning cell was flushed with cold N2 gas and temperature was measured with a thermocouple at the scattering point. Raman shifts were calibrated with CC4. Uncertainties of peak positions were f l cm-1 for the original spectra but *2 cm-1 for difference spectra, unless otherwise stated in figure captions. Cytochrome c oxidase (cytochrome c: oxidoreductase, EC 1.9.3.1, CcO) was isolated from bovine heart according to Yoshikawa’s method.24 About 1 mL of the 50 pM CcO solution
The Journal of Physical Chemistry, Vol. 98, No. 26, I994 6653
~
in 50 mM sodium phosphate buffer, pH 7.2, was transferred to an airtight spinning cell. The CO adduct of CcO (CcOCO) was formed through repeated evacuation of gas inside the cell (to ca. 0.1 mmHg) followed by incorporationof CO to it (to ca. 1 atm), for three times. Finally, for complete removal of oxygen, a small amount of N2-saturated dithionite solution was added so that its final concentrationwas 10mM. The CO-isotope adducts of CcO were obtained from the CcOJ2C160 sample by evacuating 12C160 and incorporating 13C160(ICON, 99 atom % for 13C), I2C18O (Cambridge Isotope Lab, 98 atom % for l80) or W 1 8 0 (Isotec, 98.6 atom % for l3C and 96.8 atom % for 1 8 0 ) . Human adult Hb (Hb A), prepared according to the method of Geraci et was separated into the constituent CY- and 8-chains by the treatment with pmercuribenzoate25 followed by fractionation with preparative isoelectrofocusing electrophoresis (pH range 6-9).% The CY- ando-chains were obtained by focusing on pZ 7.65 and 6.35, respectively. Mercury was removed from the chains by incubatingwith dithi~threitol.~~ The concentration was adjusted to 50 pM (heme) with 50 mM sodium phosphate buffer, pH 7.2, or with 50 mM Tris-HC1 buffer, pH 8.5, while in the experiments for examining temperature dependences the buffer contained 20% (v/v) glycerol. To replace the solvent H2O with D20, 3 mM Hb solution was diluted with D2O (99.75%) buffer to the concentration of 50 pM, and CO adducts were formed in the same way as described for CcO. Horse Mb (Sigma, type M630) was dissolved in 50 mM TrisHCl buffer, pH 8.5. The concentrated metMb solution was reduced by sodium dithionite and subjected to gel filtration through SephadexG-25 column. Mb02 thus obtainedwas diluted to 30 pM with the Tris-HC1 buffer, pH 8.5, and transferred to the spinning cell, and CO adducts were formed in the same way as described for CcO. Pseudomonas putida P-450 (P-450,,) was a kind gift from Drs. T. Egawa and Y.Ishimura of Keio University and its CO adducts (P-45O,,-CO) were prepared in a way similar to CcO.CO.
Results The RR spectra in the 600-200-cm-l region for l2Cl60(A) and 13C18O (B) adducts of Hb A and their differencespectra (C) excited at 406.7 nm are shown in Figure 1. The Raman bands of Hb12C160at 578 and 505 cm-I are shifted to 558 and 492 cm-I, respectively, with Hb13C180and their difference spectrum clearly demonstrating this. This is in agreement with the spectra reported so far.’ However, we note that there is a small differential pattern around 365 cm-I, which is so small that other investigators have overlooked it hitherto. Since there are relatively strong porphyrin bands around 340-380 cm-l, the presence of a COisotope-sensitive band around 365 cm-l has been difficult to notice in the raw spectra. Furthermore, if the two independent Raman measurementsfor CO-isotopederivatives includea small amount of shift in the wavenumber axis on accountof different dispositions of the spinning cell of fluctuation of the laser beam, differential patterns would appear in the difference spectra particularly at peak positions of the raw spectra. In this study special attention was paid to this problem, and the lack of a differential pattern in the difference spectrum for the plasma lines marked by an asterisk demonstratesthe reproducibility of the wavenumber axis. The differential pattern around 365 cm-l is definitely larger than the noise level in Figure 1. Figure 2 shows the same pair of RR spectra which were excited at 427.0nm. This excitation wavelength iscloser to theabsorption maximum of HbCO (419 nm), but RR spectra excited in such proximity to the absorption maximum have never been reported. The RR bands are generally more intense with 427.0-nm than 406.7-nm excitation, but we note here that the intensity of the differential pattern at 577 cm-1 relative to that at 365 cm-I differs significantly between Figures 1 and 2. The relative intensity of
6654 The Journal of Physical Chemistry, Vol. 98, No. 26, 1994 492 50d
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Raman Shift / cm" Figure 1. RR spectra in the 600-200-~m-~ region of the I2CL6O (A) and W 8 O (B) adducts of Hb A and their difference (C). The ordinate scales in spectra A and B are normalized with the intensity of porphyrin bands. Peaks marked by an asteriskdenote the plasma lines from a Kr+ ion laser. Experimentalconditions: excitation, 406.7 nm, 6 mW at the sample;detector, diode array; sample, 50 pM (heme) in 50 mM sodium phosphate buffer, pH 7.2. An arrow for spectrum C indicates the difference pattern around 365 cm-I. the 577-cm-' to the 505-cm-1 band in the raw spectra is reduced on excitation at longer wavelengths. Figure 3 displays the RR spectra in the 600-200-~m-~region of W 6 0 (A), W'6O (B), 12C180(C), and 13C1*0(D) adducts of H b A excited at 426.0 nm. The strong band at 505 cm-1 in spectrum A shifts monotonously to lower frequencieswith increase of the total mass of CO, that is, to 501 cm-1 with 13C'60,496 cm-' with IZC180, and 492 cm-' with 13C18O. Therefore, this is assigned to the Y F ~ mode O as believed so far.' The presence of a porphyrin band at 587 cm-' often makes it difficult to estimate correctly the intensity of the CO-isotope-sensitive band around 575 cm-1. The CO-isotope-sensitive band is located at 576 cm-' in Figure 3A, which shifts to 561 cm-' with I3CI6O,to 574 cm-' with W * O , and to 558 cm-1 with 13C180.These frequencies are in agreement with thereported values.'" Since it exhibits a zigzag pattern against the increase of the total mass of CO, it has to date been assigned as 6 ~ ~ ~ 0In.the 1 frequency region below 500 cm-I, it is apparently difficult to see similar CO-isotopic frequency shifts in the raw spectra. In fact, the C&Cd bending bands of the propionic side chain28 at 385 and 373 cm-' remain unchanged with the CO isotopes, but we note that the left wing of a broad band around 355 cm-' is definitely influenced by the CO isotopes. In order to clarify the changes contained, difference spectra with regard to the 12C160adduct were calculated. The results are illustrated in Figure 4 where positive peaks at 577,506, and 367 cm-' common to all three spectra are assigned to the W 1 6 0 adduct. It is seen that the differential feature around 575 cm-1 is appreciable when a carbon atom was replaced by an isotope but is extremely weak when an oxygen atom was replaced by an isotope. We emphasize that there is a CO-isotopesensitive band around 365 cm-' which exhibits the same character as the 575-cm-' band; Le., the intensities of the difference peaks are larger for carbon replacement than for oxygen replacement, and their frequencies exhibit a zigzag pattern against the total
Hirota et al.
I HUMAN HbCO
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Raman Shift I cm-' Figure 2. RR spectra in the 600-200-cm-1region of the l2CL6O(A) and 13C180(B) adducts of Hb A and their difference (C). The ordinate scales are treated in the same way as that in Figure 1. Experimental
conditions: excitation, 427.0 nm obtained with a Ti-sapphire laser, 4 mW at the sample; detector, diode array; sample, the same as that for Figure 1. HUMAN HbCO
0
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Raman Shift I cm-' Figure 3. Comparison of RR spectra of the four CO-isotopeadducts of Hb A in the 600-200-~m-~region. The ordinate scales are normalized
with the intensity of porphyrin bands. Experimentalconditions: excitation, 426.0 nm obtained with a Ti-sapphire laser, 5 mW at the sample; sample, 50 pM (heme) in 50 mM Tris-HC1 buffer, pH 8.5. mass of CO. The difference-peak pattern around 365 cm-' displays slight asymmetry, its trough being shallower and broader than the symmetric case. In order to see if the 365-cm-' band
The Journal of Physical Chemistry, Vol. 98, No. 26, 1994 6655
FeCO Bending Fundamental of Heme Proteins
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c o - 13c18o 427.0 nm 353
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Raman Shift / cm” Figure 4. Differences between the RR spectra shown in Figure 3. The differenceswith regard to the 12C160adduCtaredisplayed. Thedifference mmbinationsarespecifiedattheleftofeachspectrum.Thepeakpositions are an average of several measurements, and their standard deviations are at most 3 cm-l. arises from a difference combination of the hot 575-cm-1 mode with an unknown mode, temperature dependences of RR spectra were examined. Figure 5 shows the RR difference spectra in the 600-300-~m-~ region of HbW160 vs Hb13C180 observed at 20 (A) and -10 OC (B). The ordinate scales of the two difference spectra were normalized by a porphyrin band of the individual raw spectra. The presence of 20% glycerol altered little the RR spectrum at 20 OC, but at -10 OC the peak positions were shifted to higher frequencies by 1-3 cm-l for the 575- and 365-cm-1 bands while the 505-cm-1 band remained unshifted. Although the relative peak intensities of the CO-isotope bands in the difference spectra are almost unaltered upon lowering the temperature to -10 OC, the difference peak intensities do not always reflect the correct intensities of raw spectra when frequency shifts are involved. The raw spectra did not allow the estimation of intensity of the 365cm-1 band. Accordingly, we failed to demonstrate the absence of the intensity change of the 365-cm-1 band by 11% which is expected for the difference combination of the excited 210-cm-l band. The difference-peak frequencies correspondingto the 575- and 365-cm-1 bands for each CO-isotope adduct in Figure 4 are summarized in Table 1. Note that the frequency separation of those two bands appeared to be less sensitive to the mass of CO, Le., 208 f 3 cm-1, but it is meaningfully sensitive to the mass of carbon atom of CO. Since this is close to the Fe-histidine stretching ( Y F + H ~ ) frequency, it was suspected that the 575-cm-l was a combination of d ~ d owith V F + H ~ , but this idea will be discarded in the experiments on P-450” as described later. Figure 6 shows the RR spectra in the 600-300-~m-~region of I T 1 6 0 (A), 13C16O (B), 12Cl80 (C) and 13C18O (D) adducts of CcO excited at 426.0 nm. The band at 516 cm-1, which was previously assigned to V F ~ ofO cytochrome a3,29 shifts monotonously with an increase of mass of C0:5 12 (13CW), 506 (12c180), and 503 cm-1 (13C180). The band of the 12CI60 adduct at 574 cm-1, which exhibits the zigzag pattern, i.e., 559 (13CI6O), 572
1
I BCX)
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Raman Shift I cm-’ Figure 5. Hb1X!160versus H b W 8 0RR differencespectra in the 600300-cm-l region at 20 (A) and -10 OC (B). The ordinate scales of the difference spectra are normalized by the intensity of porphyrin bands in individual raw spectra. Experimental conditions: excitation, 427.0 nm obtained with a Ti-sapphire laser, 1.5 mW at the sample; detector, diode array; sample, 75 pM (heme) in 50 mM sodium phosphate buffer containing 20% glycerol, pH 7.2.
TABLE 1: Peaks in the Difference Spectra for the b a and Combination Modes and the Frequency Differences of These Peaks for Each CO-Isotope Adduct of Hb. bF&Ob combination difference lZC160 367 577 210 13C160 355 56 1 206 12C180 360 571 21 1 13C180 353 558 205 Average frequencies (in cm-l unit) in several measurements. The peak positions for the 355-,360-, and 353-cm-l bands have the standard deviation of 3 cm-l, but others have that of 2 cm-1. (12C180),and 556 cm-l (13ClsO), has to date been assigned to These frequencies for the I2Cl6Oand 13C160adducts are in agreement with the reported values,Z9 but the others are new data. There are three porphyrin bands in the region between 330 and 380 cm-1, and their apparent peak positions scarcely change with the isotopes of CO, although the band shapes are slightly different. Differences between the four spectra shown in Figure 6 are calculated in Figure 7, where the difference spectra with regard to the W 1 6 0 adduct ( A X ) similar to Figure 4A-C and the difference between l2C180and 13C180(D) are displayed. It is evident that there is a differential feature around 360 cm-l in addition to the difference peaks at 575 and 517 cm-1, but more than one set of positive and negative peaks are seen in the 360cm-1 region of Figure 7A-C. In order to explore the excitation wavelength dependence of the CO-isotope-sensitive bands, the RR spectra of the l2C*60 and 13C18Oadducts of CcO were measured at severalwavelengths near the Soret maximum of cytochrome arCO. Their difference spectra are illustrated in Figure 8, where the peak intensity of the Y F ~ band O at 516 cm-1 is adjusted to be common in all the difference spectra. The two sets of positive and negative bands in the region from 380- to 330-cm-1change in intensity with the dFd0.29
Hirota et al.
6656 The Journal of Physical Chemistry, Vol. 98, No. 26, I994
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Raman Shift / cm” Figure 6. RR spectra of Cc0.CO in the 600-300-~m-~region for l2CI6O (A), I3Cl6O (B), 12C180(C) and l3C18O (D) adducts. The ordinate scales of the four spectra are normalized by the intensity of porphyrin bands. Experimental conditions are the same as those for Figure 3 exccpt that the laser used was a dye laser and that the sample condition was 50 pM (heme) in 50 mM sodium phosphate buffer, pH 7.2.
’
Ill 1
Figure 8. Excitation wavelength dependence of the difference spectra between CcOJ2c160 and CcOJ3C180. Difference calculations were carried out in the same way as those for Figure 4. Experimental conditions: laser, 1 mW for ( A X ) and 5 mW for (D) generated with a dye laser; detector, diode array; sample, 50 pM (heme) in 50 mM sodium phosphate buffer, pH 7.2.
BOVINE CCOCO 426.0 nm
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Raman Shift / cm” Figure 7. Differences between the RR spectra of CcO.CO shown in Figure 6. Differences with regard to the 12C160adduct are shown in spectra A X while the difference between the 12C180 and 13C180adducts is shown in spectrum D. The difference combinations are specified at the left of each spectrum. excitation wavelength, suggesting that they share intensity via vibrational coupling, but this will be discussed later. Figure 9 shows the RR spectra in the 8 0 0 - 2 0 0 - ~ m - ~region of
1
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Raman Shift / cm“ Figure 9. RR spectra in the 800-200-~m-~ region of P-45O-CO in the presence of 1 mM camphor. (A) 12C160,(B) 13C180,(C) 12C180,(D) W 8 O . The ordinate scales of four spectra are normalized with the intensity of porphyrin bands. Experimental conditions: excitation, 441.6 nm, 5 mW at the sample; sample, 40 pM in 50 mM sodium phosphate buffer, pH 7.2. l*CI*O(C), a n d Wl8O (D) adducts of in t h e presence of camphor excited at 441.6 nm. T h e
12C160 (A), 13C16O (B),
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FeCO Bending Fundamental of Heme Proteins
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The Journal of Physical Chemistry, Vol. 98, No. 26,1994 6657
P=WI,,,,,(+C~~)CO 441.6 nm
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Raman Shift / cm4 Figure 10. Differences between the RR spectra shown in Figure 9. Difference with regard to the 12c160adduct as specified at the left are displayed.
strong band at 484 cm-l in spectrum A, which was previously found at 477 cm-' and assigned to VFin ref 3a, shifts to 480 cm-1 with W 6 O , to 475 cm-1 with 1zC180,and to 472 cm-1 with 13C180. Although the frequency is appreciably lower, the extent of frequencyshiftof the 484-cm-l band on CO isotopicsubstitution is the same as those of Hb A (Figure 3). The other CO-isotopesensitive band is seen at 559 cm-1 for W160,546 cm-1 for 13C160, 557 cm-' for lfC180, and 544 cm-' for 13C180. The similarity in the isotope shifts suggests that the 559-cm-l band of P-45O,,.CO arises from the same modes as the 576-cm-l band of HbCO and the 574-cm-1 band of CcOCO. Since there are porphyrin bands at 378 and 353 cm-I, it is not clear in Figure 9 whether a CO-isotope-sensitive band is present around 350 cm-1 or not. Their difference spectra with regard to the 12C160adduct are shown in Figure 10, where the porphyrin band at 674 cm-l has been completely subtracted. For spectrumB the differencepattern of the 560-cm-l band is extremely weak in comparison with that of the Y F ~ band O and similarly the difference feature around 360 cm-I is obscure. However, the presence of the CO-isotopesensitive band around 365 cm-l is evident in spectrum C. The frequency difference between the 575- and 365-cm-1 bands is 201 and 204 cm-1 for the l * C Wand 13C180adducts, respectively. This value is quite different from the Fe-S (proximal cysteine) stretching frequency around 350 cm-1.30 Consequently, it is unreasonable that the 575-cm-1 band is a combination of the d ~ e omode with the Fe-fifth ligand (trans) stretching mode. Figure 11 shows the RR spectra in the 6OG200-cm-1 region of the I2Cl6O(A) and W 8 O (B) adducts of horse Mb excited at 406.7 nm and their difference (C). Similar to other proteins, MbCO exhibits strong CO-isotope-sensitive bands around 575 and 510 cm-1, although the positive peak around 575 cm-1 in spectrum C actually consists of two peaks at 583 and 575 cm-1. There is a shoulder at the high-frequency side of the 494-cm-1 band of the W18O adduct (spectrum B). Since this spectrum does not show any feature of a band at 575 cm-1, contamination by a W 1 6 0 adduct is unlikely. This rather means that there is a porphyrin band around 505 cm-' for MbCO and it is overlapped in spectra A and B.
i 0
Raman Shift / cm" Figure 11. RR spectra of l2c160 and 13C1*0adducts of horse Mb and their difference spectra. The ordinate scales of the four spectra are normalizedwith the intensityof porphyrin bands. Traces C and F indicate the difference;spectrum C = spectrum A spectrum B and spectrum F = spectrum D - spectrum E. Experimentalconditions: excitation, 406.7 nm and 5 mW (at the sample) for (A) and (B), and 426.0 nm obtained with a dye laser and 1 mW (at the sample) for (D) and (E); detector, diode array; sample, 30 rM in 50 mM Tris-HC1 buffer, pH 8.5.
-
It is quite unexpected that spectrum C exhibits no differential features in the lower frequency region. This was also the case with the CO adduct of sperm whale Mb (data not shown). When excitation wavelength was shifted to longer wavelengths (426 nm), the intensity of the 575-cm-1 band relative to the Y F ~ O band at 509 cm-I reduced significantly as shown by spectrum D. Spectrum F in Figure 11 illustrates the difference between the RR spectra of the same isotope species as (A) and (B) excited at 426.0 nm. The differential pattern around 575 cm-1 is significantlyreduced in intensity comparedwith that of the Y F ~ O band, but again there is no differential feature in the 365-cm-1 region. In order to get an insight into the difference between Hb and Mb, the isolated chains of Hb, which have been postulated as very similar to Mb," were investigatedand the results are shown in Figure 12, where panels a and b display the spectra of the aand @-chains,respectively. There are appreciable differences between the a-and 8-chains in the raw spectra, but the difference spectra of their W60and 13C180adducts shown by spectra C are similar. The intensity of the 575-cm-l feature relative to that of Y F ~ isO much weaker with Hb chains than with Mb (Figure 1 1F). However, contrary to Mb, the isolated chains of Hb A give rise to the differential pattern with a peak at 367-368 cm-1 and a trough a 351-353 cm-1. In this regard, Hb and Mb are dissimilar. Figure 13 shows the effects of deuteration of globins of Mb (a and b) and Hb (c and d) on the CO-related frequencies. All were excited at 406.7 nm. Here the spectra of Hz0 (A) and DzO (B) solutions and their difference (C) are displayed for the 12C160 (a and c) and 13C180(b and d) adducts. It is stressed that the 575-cm-' feature exhibits a high-frequency shift in DzO while the Y F ~ band O is scarcely shifted and that the 575-cm-1 band of Mb undergoes more intensity enhancement in D20. In the caseof MbCO, a porphyrin band at 584 cm-' seems to be coupled
6658 The Journal of Physical Chemistry, Vol. 98, No. 26, 1994
(A) 12C160
(B)
l3cl8o
s
6
k
i
-
m
Raman Shift I cm"
Raman Shift I cm" Figure 12. RR spectra in the 600-200-~m-~region of the 1zCL60and W80adducts of the a- and @-chainsof Hb A. Panels a and b show the results for the a- and @chains, respectively, and traces A and B denote the spectra of the l2C160and l3Cl80 adducts, respectively. Traces C indicate the difference between spectra A and B. The ordinate scales are normalized with porphyrin bands. Experimental conditions: excitation, 427.0 nm obtained with a Ti-sapphire laser, 3 mW at the sample; detector, diode array; sample 50 pM in 50 mM Tris-HC1 buffer, pH 8.5.
H20 D20 (C) A B
-
584
I
600 550 500 450 600 550 500 450 Raman Shift I cm" Raman Shift I cm" 406.7 nm
(C) A
406.7 nm
--
-B
579
H
581
W
600 550 500 450 600 550 500 450 Raman Shift I cm" Raman Shift / cm"
Figure 13. RR spectral differences between H20 and DzO solutions of C O adducts of Mb and Hb. (A) and (B) represent the spectra observed for the H20and DzOsolutions, respectively,and (C) shows their difference; (a) MbITl60, (b) MbI3CCL80, (c) HblZCl60, (d) Hbl3CI80.'Theordinate scale of each spectrum is adjusted so that the intensity of the V F ~ band O is the same. Peaks marked by an asterisk denote the plasma lines from a Kr+ ion laser. Experimental conditions: excitation: 406.7 nm, 5 m W at the sample; detector, CCD for (a) and (b) and diode array for (c) and (d); sample, 30 pM for MbCO and 50 pM for HbCO both in 50 mM sodium phosphate buffer, pH 7.2.
with the 577-cm-1 band in H2O and it appears as a doublet, whereas this coupling is removed in D20. This is reflected in the differencespectrum as a negative doublet (C in panel a). When the frequency is shifted by using 13C180,the vibrational coupling becomes less important and the band appears as a singlet in both H20 and Dz0, and its frequency is higher in D2O than in H20.
Hirota et al. Since its intensity is increased in DzO, the difference spectrum (C in panel b) does not display a typical differential pattern when the v ~ dband o is adjusted to disappear. The correspondingbands of HbCO also display shifts to higher frequencies in DzO (C in c and d), indicating that the vibrational properties of the 575cm-1 bands of HbCO and MbCO are alike. The deuteration shift to higher frequenciescannot be explained by a simple effect of a hydrogen bond between CO and E7-His on the b ~ d bending o mode, since such a frequency shift would also be expected for the vco and V F stretches ~ (except in the special case of a hydrogen bond nearly perpendicular to a linear Fe-C-O unit). Such a frequency shift on deuteration is not found for the stretching modes. This difference in behaviors between the 575-cm-I and the stretching bands makes it unlikely that the 575-cm-l band is the fundamental of the FeCO bending mode. However, we were unable to determine if there was a deuteration shift for the 365cm-1 band due to its weakness.
Discussion The FeCO Bending Fundamental Mode. It became evident from this study that there is a CO-isotope-sensitive band around 365 cm-1 in addition to the bands around 575 and 500 cm-I for CcO.CO, P-45O,,.CO, HbCO, and CO adducts of the isolated a-and j3-chains of Hb A. Since the assignment of the 500-cm-I band to V F ~ isOof no doubt, either the 575- or 365-cm-l band should be the d ~ d fundamental o and the other a nonfundamental mode. Generally, the intensity of the difference combination band becomes significantly weaker when temperature is lowered due to depopulation at an excited level. If the 365-cm-l band were a difference combination of a hot porphyrin mode around 210 cm-l with the 575-cm-I mode, its intensity would be expected to decrease by 11% upon temperature decrease from 20 to -10 "C. Since the 365-cm-I band exhibited a small frequency shift upon lowering temperature, it is quite difficult to determine from differencespectra A and B in Figure 5 if this amount of intensity changeisinvolvedbetween20and-10 OCor not. Thisexperiment alone cannot determine it, but all the data collected so far suggest that the assignment of this band to the difference combination seems less plausible. In the excitation profile measurements,the 575-cm-I band has the maximum of intensity enhancement at shorter wavelengths than that of the 365-cm-1 band as seen in Figures 1,2,8, and 11. In RR scattering, the intensities of overtone and combination bands relative to those of the fundamental become stronger as the excitation wavelength is brought to shorter wavelength^.^^ Therefore, the excitation profile data favor that the 575-cm-1 band is a combination and the 365-cm-1 band a fundamental rather than the reversed assignment. Furthermore, the FeCO bending frequency is lower than the Fe-C stretching frequency for CO adsorbed on an Fe surface.33 For 02 adducts of Hb and CcO, the Fe-O-O bending RR band was recently observed around 430 cm-1,34which is also significantly lower than the Fe-02 stretching frequency (570 cm-l). These facts also support that the 365-cm-1 band rather than the 575-cm-1 band isa fundamental of GFcCO. The main justification for assigning the 575-cm-1 band to a 6 ~ d fundamental o was the zigzag pattern of its frequency against the increase of the total mass of CO. The 365-cm-1 band exhibits the same feature. As a result, the frequency differencesbetween the 575- and 365-cm-1 bands are nearly constant among different CO-isotope adducts of Hb as shown in Table 1. Furthermore, the absolute values as well as their isotopic frequency shifts of the three bands can be well reproduced by a simple normalcoordinate calculation for the isolated FeCO unit. In the same way as our previous treatment? the potential function (V) represented by eq 1 was used,
2v= ~~b~~~~ + ~~b~~~ + HA^,,^ + FA^,'
(1)
FeCO Bending Fundamental of Heme Proteins
TABLE 2 Calculated Frequencies for the Isolated FeCO Unit and Observed Freauencies of HbCW IF160 IC160 W 8 0 130180 ~
Calc ob Calc obs Calc obs Calc 1862 1861 1906 1908 1952 1907 1907 vco 1952 496 492 489 506 501 499 496 V F ~ O 505 6peo 367 366 355 356 363c 360 353 350 a In cm-l unit. b Parameters used for calculations are r(Fe-C) = 1.8 A,r(C-O) = 1.2A,&(Fe-C) = 2.05mdyn/A,K2(C-O) = 14.62mdyn/ A, H ( F 4 - O ) = 0.38 m d y d , F(Fe-0) = 0.7mdyn/A, B(Fe-C-0) = 1 6 5 O . Since the frequency of the negative peak corresponding to the 574-cm-1 band is read lower by 3 cm-1 in Figure 4B than that in Figure 3C, the reading for the 365-cm-1 band in the difference spectra (Figure 4B) was deduced to be higher than the true frequency by 3 cm-1, and therefore the frequency is estimated to be 363 cm-l. obs
where b ~ d ob,c o . A ~ F ~and o , hr~a denote the displacement coordinates for the Fe-CO stretching, C-O stretching, Fe-C-O bending, and F e O nonbonding interaction, respectively. For a suitable bending angle as seen for Hb (6= 165"), force constants were adjusted so as to reproduce the observed frequencies of HbCO under the assumption that the 365-cm-I band is the fundamental of 6 ~ d 0 . The calculated results are compared with the observed values in Table 2, where the values of force constants are also cited. The validity of the force constant of H = 0.38 mdyn.A is unknown since there are no ab initio calculations available. Spiro and co-workers used H = 0.8 mdyn.A5a.2l and justified the frequency of higher than U F ~ byO referring to data on Mn(C0)sBr. However, Jones et used 0.45,0.48, and 0.48 m d y d for the Fe-C-O bending force constant of the valence force field in their normal-mode calculations of Mo(CO)~, Cr(C0)6, and W(CO)6, respectively. It is apparent that the electronic state of Fe in Fe-porphyrin is quite different from that of Mn of Mn(CO),Br and thus the m e t a l 4 0 bending force constants could be significantly different. Therefore, the magnitude of the Fe-C-0 bending force constant cannot be used as a diagnosis of correct assignment as the present stage. As far as thezigzag patternoftheisotopic frequency shift ofCO, the present assignment is reasonable, although a more refined force field is required to reproduce the observed frequencies precisely. 6 ~ e Band o of CcO. The difference spectral patterns in the 365-cm-l region were clear for Hb A and P-450,, but somewhat complicated for CcO, which has heme A as the CO binding site instead of heme B for the former two. In Figure 8 there were two positive peaks at 369 and 359 cm-l for the 12C160adductand two negative peaks at 352 and 332 cm-I for the 13C180adduct, and the trough at 332 cm-' became deeper than that at 352 cm-l as the excitation wavelength became longer. It is evident from Figure 6A that the heme A C O has a porphyrin mode at 339 cm-l and this band became more intensewhen the excitation wavelength was changed from 422.5 to 435.0 nm (data not shown). If there is a porphyrin vibration with an intrinsic frequency of up, which interacts with the Fe-ligand mode having an intrinsic frequency of UL, the observable frequencies would be U L =~V L 6 and v p o b = up - 6 if U L > up or V L O ~= UL - 6 and upok = UP 6 if U L < up. The size of 6 depends on the magnitude of the interaction and separation between Y L and up modes. For the W18O adduct of CcO, if we assume that u p = 339 cm-l and vpok = 332 cm-I, it means 6 = 7 cm-1 and therefore = 353 cm-I would yield U L = 346 cm-1. The values of Y L for other CO isotopes can then be estimated from equations mentioned above and are listed in Table 3 together with the values of up and 6 assumed. For the 12C'80/'3C180difference spectrum, a single set of upob" and U L O is ~ seen in the difference spectrum (D in Figure 7) since their counterparts apparently disappear due to subtraction. However, it cannot be reasonably explained why 13C160exhibits no splitting despite the proximity of V L to u p = 339 cm-1. In addition, for the 13Cl*Oadduct, the calculated separation between
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The Journal of Physical Chemistry, Vol. 98, No. 26, 1994 6659
TABLE 3 Observed Frequencies and Estimated Intrinsic Fe-C-0 Bending Fundamental Frequencies for CO-Isotope Adducts of Cytochrome c Oxidasp observedb estimatedc combination v p h vp VL 6 difP 12C160 574 356 368 -358 366 2 208 559 343 (343) 0 216 13C160 12cl80 572 360 352 -358 354 2 218 l3cl80 556 353 332 -339 346 7 210 Incm-l unit. * Frequencicsarereadfromthedifferencespectrashown in Figure 7. vp are assumed and YL are deduced according to eq 1, V L ~ , and 6. d Difference between the combination mode and VL. and UL is too large to yield 6 = 7 cm-'. It might imply that is an out-of-plane mode buried in the strong in-plane bands in Figure 6 or that more than two modes are coupled. Consequently, the estimated YL wouldcontain large errors. Nonetheless, we note that the differences between the observed combination band and the inferred UL fall within 213 f 5 cm-1, which is close to the corresponding values of HbCO shown in Table 1. If there were no 6 ~ fundamental a in this frequency region, thevibrational coupling with a porphyrin mode would be so small that no COisotope-sensitive RR band would be observed around 350 cm-'. Assignment of the 575-cm-l Band. If the bound CO adopts an upright geometry against the porphyrin plane, 6~e0 is doubly degenerate and is IR active but RR inactive. When CO is slightly deviated from this structure, the degeneracy of is removed; one is pure bending and the other becomes an internal rotation around the Fe-C bond. In addition, there exists a Fe-C deformation mode which would correspond to the freedom of rotation of the whole FeCO unit for an isolated FeCO molecule and involves the displacement of the carbon atom of CO parallel to the porphyrin plane. This mode would belong to the E species in the putative linear upright geometry (Cb)and might be coupled with Group theoretically, the combination between the split components of the degenerate FeCO bending modes or between the FeCO bending and the Fe-C deformation modes can be a candidate for the 575-cm-I band. The frequency for the internal rotation around the Fe-C bond has never been reported. Although the frequency around 210 cm-l might not be unreasonable as such a mode, it should be accompanied with an isotopic frequency shift for l*O substitution. Since the difference ( ~ 2 1 0 cm-l) between the 575 and fundamental shown in Table 1 scarcelydepends on the mass of oxygen,the Fe-C internal rotation mode is less likely as a candidate for a counterpart of the combination to yield the 575-cm-1 band. However, since the l3C isotope dependenceis appreciable for the 2 10-cm-l mode in Table 1, the Fe-C deformation mode remains as a candidate for the counterpart. Thecorresponding frequency differences between the 575- and 365-cm-I bands for 12C160,13C160, and 13C180 adducts of P-450,, are 201, 207, and 204 cm-l in Figure 10, respectively. Although these values are somewhat smaller than those for Hb, the counterpart which is combined with 6 ~ d oto yield the 575cm-1 band would be located around 200-210 cm-1. The similarity between P-450,, and Hb rules out the idea of assigning the counterpart of the combination band to a stretching or bending mode of the Fe-fifth ligand. When the direction of the CO bond is only slightly deviated from the heme normal, the FeCO bending vibration would be accompaniedby an in-planemovement of the iron atom and would be coupled with such a porphyrin mode. The vibrations involving in-plane displacements of iron ion are doubly degenerate (E) under C, symmetry. Its combination with 6 ~ e ounder Cb symmetry will include RR active species: E X E = AI A2 + B1 B2. Accordingly, it is group theoretically reasonable that the fundamental is very weak but the 575-cm-1 band has appreciable intensity. However, it is necessary to explain why the particular porphyrin mode is deuteration sensitive. Possible up up
+
+
6660 The Journal of Physical Chemistry, Vol. 98, No. 26, 1994 differencesin the strength of hydrogen bonds between protonated and deuterated proteins may influence some low-frequency porphyrin vibrations. If the effective symmetry is lower than C,, group theoretical consideration is not helpful. It is desirable to specify the counterpart of the combination by observing IR spectra for model compounds, and such a study is under progress in this laboratory. The deuteration shift of the 575-cm-1 band is puzzling. Since this is an upshift, it would not be caused by a simple mass effect. In the Of the Fe'V4 heme in "pound 'I Of horseradish peroxidase, the F e I V 4stretching frequency was shifted to a higher frequency in DzO due to weaker deuteron than hydrogen bonding to the bound o ~ y g e n However, . ~ ~ ~ ~ IR ~ measurements of vco on HbCO and CcO.CO noted no detectable effects of protein deuteration on the YCO frequen~y.3~ The ~ ~frequency 4 0 is also insensitive to deuteration as demonstrated in Figure 13. It is unlikely that a putative hydrogen bonding to CO causes a frequency shift only for the Fe-C-0 bending but not for the Fe-CO and C-0 stretching except for a less likely structure in which the,hydrogen bond to the oxygen atom is perpendicular to the C-0 bond. Therefore, the deuteration shift data are also unfavorableto assign the 575-cm-*band t o t h e h d u n d a m e n t a l . If the counterpart of 8 ~ a in o the combination mode were slightly altered by protein deuteration, the 575-cm-1 band would also be
so. Hb and Mb are oxygen-carrier proteins having evolutionally a common origin of genes. Both have a histidine coordinated protohemeand another histidine near the ligand-bindingsite called the distal histidine. The oxygen-binding affinities of the isolated a- and 8-chains of Hb A are close to that of Mb.3' The angle between the CO axis and the heme normal, determined by picosecond IR spectroscopy' is 180 for HbCo and 2oo (main component, vco = 1940 cm-l) for sperm whale MbC0.39 These facts have led one to think that the surrounding structures around the ligand binding site of Hb and Mb are alike. However, the results shown in Figures 11-13 revealed distinct differences between the CO adducts of Hb and Mb. Although thevibrational characters are little altered between Hb and Mb, the very strong intensity of the 575-cm-1band, its larger upshift in DzO, and the absence of the 365-cm-1 band are characteristic of Mb. Such differences are also seen in 02 addcuts for which the Fe-0-0 bending RR band is observable for HbO2 but not for Mb02.34 The origin of these differences remains to be explored. This would clarify the band origin of the 575-cm-1 band. Acknowledgment. The authors wish to express their gratitude to Drs. T. Egawa and Y.Ishimura of Keio University for their courtesy of giving them purified P-450,,. They also thank Prof. P. Stein of Duquesne University and Dr. P. Jewsbury of Institute for Molecular Science for stimulating discussions. This study was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture, Japan for Priority Areas (bioinorganic chemistry, 03241105) to T.K. References and Notes (1) (a) Kerr, E.; Yu, N. T. In Biological Application of Ramun Spectroscopy; Spiro, T. G., Ed.; Wiley-Interscience: New York, 1988;Vol. 3, pp 39-95. (b) Yu, N. T. Methods Enzymol. 1986,130, 350409. (2) (a) Alben, J. 0.;Caughey, W. S.Biochemistry 1968,7 , 175-183. (b) Caughey, W. S.;Alben, J. 0.;McCoy, S.;Boyer, S.H.; Charache, S.; Hathaway, P. Biochemistry 1969,8,59-62. (3) (a) Tsubaki, M.; Ichikawa, Y. Biochim. Biophys. Acta 1985,827, 268-274. (b) Tsubaki, M.; Hiwatashi, A.; Ichikawa, Y. Biochemistry 1986, 25,3563-3569.
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