THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 6, lasue of March 25, pp. 351%3528,1985 Printed in U.S.A.

Investigations of Cyanide as an Infrared Probe of Hemeprotein Ligand Binding Sites*

(Received for publication, August 6, 1984)

Shinya YoshikawaS, David H. O'KeeffeO, and Winslow S . Caughey From the Department of Biochemistry, Colorado State University, Fort Collins, Colorado 80523

The measurement of infrared spectra for cyanide liganded to hemeproteins and hemins has been inves- tigated. The hemeproteins included human methemo- globin A, lamprey methemoglobin, metchlorocruorin, horse metmyoglobin, and horseradish peroxidase. The hemins were dicyanide and monopyridine monocya- nide species of deuteroporphyrin IX iron(II1) and its Z,.l-divinyl(proto) and 2,4-diacetyl derivatives. C-N stretch bands of low intensity detected near 2100 cm-' exhibit changes in frequency, width, intensity, and isotope shift with changes in cyanide compound struc- ture. Infrared band parameters are particularly sen- sitive to a change in oxidation state (Fe2+ uersus Fe3+) and are affected to a lesser extent by changes in por- phyrin ring substituent, ligand trans to the cyanide, and protein structure. Evidence of multiple conformers (Le. multiple C-N stretch bands) was found for several hemeproteins. The cyanide infrared spectra provide direct evidence for cyanide binding as a metal cyanide (FeC=N) and against HCN being the ligand in ni- trile-like bonding ( F e N a - H ) in all the hemepro- tein and hemin cyanides studied. With the reduced horseradish peroxidase cyanide, differences between infrared spectra for D20 and HzO solutions can re- sult from hydrogen bonding between a protein amino acid residue and the distal atom of the cyanide (Fe-C"'U- *H+-R). The binding of cyanide to re- duced iron (Fez+) of a hemeprotein was only observed in the case of the reduced peroxidase. These findings demonstrate that cyanide infrared spectra can not only determine when cyanide is bound to a metalloprotein but can also provide information on how the cyanide is bonded to metal and on characteristics of the ligand binding site.

Cyanide and carbon monoxide are widely used as ligands in investigations of those sites on metals in heme and other proteins that are accessible to external ligands. CN- and CO have long been of particular interest as classic inhibitors of respiration (1, 2). CO competes with, and mimics, oxygen binding to reduced iron (Fe2') but does not bind to oxidized iron (Fe3+) in hemeproteins. The resulting hemeprotein car- bonyls contain low-spin iron(I1) and are generally more stable than either the oxy or deoxy hemeprotein complexes. Infrared spectra of the carbonyl ligand have proven highly useful for

* This work was supported by United States Public Health Service Grant HL-15980. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Permanent address: Department of Biology, Konan University, Okamoto, Higashinada-Ku, Kobe 658, Japan.

Present address: Department of Chemistry, University of Mich- igan at Flint, Flint, MI 48503.

the characterization of oxygen binding sites (3,4). In contrast, cyanide binds with high affinity to many oxidized (Fe3') hemeproteins but with low affinity, or not at all, to reduced (Fe2+) species. Cyanide has been widely used to prepare stable, low-spin iron(II1) hemeproteins. However, infrared spectros- copy of bound cyanide has not been explored in detail. McCoy and Caughey (5) have reported the observation of C-N stretch bands for metMbCN and metHbCN near 2125 cm" which is within an absorption window in both H20 and D20 (3). Cyanide was found to have some disadvantages as an infrared ligand probe compared to CO, e.g. the C-N stretch bands are much less intense than C-0 stretch bands and bands for unliganded cyanide as free anion or HCN appear in the same region of the spectrum as the cyanide that is bound to heme iron. However, cyanide has an important advantage of being able to bind to iron(III), whereas CO cannot. If achievable experimentally, the direct measurement of the vibrational spectrum of cyanide bound to a hemeprotein could undoubt- edly provide uniquely important information about the bind- ing interactions between the ligand and its binding site.

We report here an investigation of the utility of infrared spectroscopy of bound cyanide in the study of iron(II1) and iron(I1) ligand binding sites in hemeproteins. C-N stretch bands for cyanide bound to horse metMb, human and lamprey metHbs, metchlorocruorin, and horseradish peroxidase (both iron(I1) and iron(II1) species) have been compared with bands for 2,4-substituted deuterohemin cyanides. Chlorocruorin, an oxygen carrying protein from a species of polychaete, contains 2-formyl-4-vinyldeuteroheme rather than the 2,4-divinyldeu- teroheme present in the other hemeproteins. Effects of sub- stitution of D20 for H20 in the medium and of I 3 C for '*C or 15N for 14N in the cyanide ligand on cyanide spectra were observed. We conclude that cyanide infrared spectra can provide highly useful information for the characterization of the bonding between the ligand and its binding site within a hemeprotein or any other metalloprotein with a metal site to which cyanide can bind.

EXPERIMENTAL PROCEDURES

Non-heme Cyanide~-K"C'~N was obtained from Baker and DzO, NaI3C"N, and Na'2C16N from Prochem. Deuterium cyanide (DCN) solutions were prepared by neutralizing KCN in DzO with DC1. Cyanide solutions were kept at 0 "C in tightly stoppered flasks and used on the day prepared.

Hemin Cyanides-Hemin B chloride (protohemin) was obtained from Eastman. Deuterohemin IX chloride and 2,4-diacetyldeutero- hemin IX chloride were prepared as described previously (6). To prepare the cyanide complexes, the hemin chloride, which was 20-50 mM in aqueous pyridine (HzO/pyridine, DzO/pyridine, or DzO/pyri- dine-d6, 1:3 (v/v)) was treated with KCN. As the concentration of KCN was increased, shifts in visible-Soret, 'H NMR, and infrared spectra were in accord with shifts in equilibria from dipyridine to monopyridine monocyanide to dicyanide species (see "Results").

Hemeprotein Cyunides-HbAOz was obtained from fresh human

3518

Infrared Spectra of Cyanide Bound to Hemeproteins 3519

blood by the method of Geraci et ul. (7). Oxychloroc~orin was isolated from sea worms (Potumilta tu) by the method of Orii et ut. (8). MetHbA and oxidized chlorocruorin were prepared from oxy species in 10 mM Na phosphate buffer, pH 7.4, by the addition of sufficient KPe(CN)6 to convert all oxy to met species as determined from visible-Soret spectra. Dialysis of the metHb solution against the same buffer for 15 h was followed by concentration to 20 mM in heme with an Amicon Diaflow apparatus. Lamprey Hb was isofated in this laboratory from fresh blood of Petromyzon murinus by Dr. Melvin Tucker; the only fraction used represented the major band eluted as the oxy species from a DEAE-Sephadex column with 5 mM Tris buffer, pH 8.5, followed by oxidation and concentration as described above. Horse metMb (a lyophilized product from Sigma) contained some MbO, and was therefore treated with ferricyanide and concen- trated as described above for the hemoglobins in order to obtain a metMb solution. Oxidized horseradish peroxidase (Sigma, Type VI) was used as obtained without further purification. Reduced horserad- ish peroxidase was obtained by addition of a slight excess of sodium dithionite to horseradish peroxidase solutions under NS. The cyanide derivatives of the hemeproteins were prepared by addition of an appropriate volume of a soiution of 0.5 or 1.0 M HCN or DCN. Conversion to the cyanide spectra was monitored by comparing visible-Soret spectra with literature values.

Recording of Spectra-Infrared spectra were measured with a Per- kin-Elmer Model 180 spectrophotometer interfaced with a Tektronix Model 4051 computer. ~ is jb~e-sore t spectra were recorded with a Cary Model 17 spectrophotometer. Variable temperature infrared cells (Beckman FH-01) with CaFz windows and a Teflon spacer 0.052 mm thick were used for the infrared and visible-Soret spectral mea- surements which were carried out as described earlier (9). Protein concentrations were determined based on the following extinction coefficient values: 9.04 mM" cm" at 500 nm for met% (IO), 10.0 mM" cm" a t 540 nm for oxidized horseradish peroxidase (111, and 6.70 mM" cm-l at 600 nm for oxidized chlorocmorin (8). For horse metMb, concentrations were determined following Na dithionite re- duction in terms of a deoxyMb extinction coefficient of 13.8 m"' cm" at 560 nm (12). Proton NMR spectra were recorded on a JEOL 100 MHz (JNM-4H-100) spectrometer operating in the field/fre- quency mode at 20 "C using tetramethylsilane as an external refer- ence.

RESULTS

Unliganded Cyanides-The free cyanide anion in either H,O or D20 exhibits a C-N stretch band at 2079 cm" (Table I). The band is rather broad (h,+ = 15.5 cm") and weak ( e = 0.03 mM" cm"). HCN in H,O and DCN in D20 exhibit C-N stretch bands at 2093 cm" and 1887 cm", respectively. Band widths of 13.0 cm" for HCN and 52 cm" for DCN are slightly less and much greater, respectively, than for the free anion. The relative amounts of free and protonated anion present depends on pH; the pK, for HCN is 9.3. The C-N stretch band parameters for the free anion are the same in aqueous alkaline buffers as in pure water, but the presence of pyridine does cause a slight reduction in uCN (Table I). Both I3C and 15N result in signi~cant isotope shifts (Table 11) with the magnitude of shift dependent upon the cyanide species.

Hexacyanoferrutes-Solutions of K3Fe(CN)6 and K4Fe- (CN), in water exhibit C-N stretch bands at 2116 and 2037 cm", respectively (Table I). Thus, the binding of six CN- anions to iron(Il.1) increases UCN by 37 cm" from the free anion value whereas the value for the six anions bound to iron(I1) is lowered by 42 cm". The oxidation state of the metal ion has a marked effect also upon the width and intensity of C-N stretch bands. The band for the iron(II1) complex is much narrower and much less intense. The ferro- cyanide anion band is comparable in width to the free cyanide anion band; both are twice as wide as the ferricyanide band. The ferrocyanide t value per CN is 4 times the ferricyanide value which, in turn, is 7 times larger than 6 for free anion.

Hemin Cyunides-The addition of increasing amounts of KCN to solutions of hemin chloride in aqueous pyridine causes changes in visible-Soret and 'H NMR spectra consist-

ent with an equi~ibrium among three hemin species (Table 111). Three C-N stretch bands are found in the infrared spectra of such solutions. When KCN is added to a mixture of water and pyridine in the volume of 1:3, respectively, the unbound cyanide anion exhibits a band with V C N = 2075 cm". However, when hemin with free (Le. unesterified) propionic acid groups are present, the band for unbound cyanide is found at 2082 cm". At low cyanide concentrations ([CN-]/[hemin] ratios less than 2), the monopyridine monocyanide band ( V C N = 2126 cm" for hemin B) is the major cyanide band. At higher cyanide concentrations, the dicyanide band (uCN = 2112 cm-I for hemin B) intensifies as the monocyanide band weakens. At [CN-]/[hemin B] ratios greater than 5, there is no band detected at 2126 cm", whereas the band at 2112 cm" is at maximum intensity. The integrated area (B value) for the 2112-~m-~ band is twice the value for the 2126-cm" band in confirmation of the assignments to dieyanide and monocya- nide species, respectively. Similar findings are found with deuterohemin IX and its 2,4-diacetyl derivative. Changes in substituent at the 2 and 4 positions of the porphyrin ring resulted in readily detected, but relatively small, changes in C-N band parameters (Table IV). The monocyanohemins consistently exhibit band widths and intensities that are similar to those for unbound CN-, whereas the dicyanides exhibit narrower bands with higher t values per cyanide ligand. No effect of substitution of DZO for HZ0 on hemin C- N band parameters was detected. The I3C and I5N isotope effects on uCN for the monocyanides of hemin B and 2,4- diacetyldeuterohemin were those expected from a reduced mass calculation (Table 11) (3).

Hemprotein Cyanides-A solution of 10.2 mM horse metMb and 42 mM HCN in 10 mM sodium phosphate buffer, pH 7.4, exhibits two infrared bands at 2126 and 2093 cm" (Fig. U).' The 2093-cm" band can be assigned to free HCN since it is found in solutions of HCN in the buffer even though metMb is absent. 13C and 15N shifts shown in Fig. 1, B and C, indicate both 2126- and 2093-cm" bands of Fig. lA are in fact due to cyanide. Furthermore, the magnitude of isotopic shifts (Table 11) are consistent with the assignment to metal bound CN- and free HCN, respectively. The effect of replac- ing Hz0 in the medium with D,O on the C-N stretch for metMb CN is too small to be considered significant (Table VI.

Support for the assignment of the 2126-cm" band to cya- nide bound to the heme iron of metMb is also found in comparisons of changes in both infrared and visible-Soret spectra as the cyanide concentration is increased in a metMb solution (Fig. 2). To obtain the data of Fig, 2, difference infrared spectra were recorded with the reference cell contain- ing 18 mM metMb without cyanide present and the sample cell containing 18 mM metMb to which neutralized KCN solution was progressively added. The visible spectral data of Fig. 2 were recorded with the same sample cell that was used for recording the difference infrared spectrum. The changes in visible spectra that accompany the conversion of aquometMb to cyanometMb are directly proportional to the increase of 2126-cm-l band intensity. Furthermore, no change

Observations while carrying out the metMbCN studies illustrated how metal cyanide impurities may also be detected in the infrared spectrum. A weak band at 2038 cm" (not shown) whose intensity varied from preparation to preparation of the Mb obtained commer- cially was obviously a contaminating metal cyanide complex. Ferro- cyanide with VCN = 2037 cm" (Table I) is the likely contaminant. Both ferrocyanide and ferricyanide exhibit strong and characteristic C-N stretch bands (Table I). Infrared s p ~ t ~ s c o p y thus provides an effective means of detecting and quantitating these frequently used hexacyanides.

3520 Infrared Spectra of Cyanide Bound to Hemeproteins TABLE I

Infrared parameters for the C-N stretch band of lZC"N in non-heme cyanides All spectra were recorded in solutions at 25 "C.

Species VCN A% c

ern" em" r n P cm" CN- Hz0" 2079 f 0.2 15.5 f 0.5 0.03 CN- DzOb 2079 f 0.2 15.5 rt 0.5 0.03 CN- Pyridine/HnO' 2075 f 0.4 14.0 rt 1.0 0.03 CN- ~ r i d i n e / H ~ O plus hemin' 2082 & 0.6 16.5 rt 1.5 d

HCN H20' 2093 & 0.3 13.0 rt 0.6 0.03 DCN DzW 1887 f 1.0 52 rt 3.0 0.04 Ferricyanide (Fea+(CN-)X-) HzOh 2116 f 0.2 8.5 f 0.4 1.18 (0.2/CN-) Ferrocyanide (Fez+(CN-)2-} H20' 2037 f 0.2 15.5 ri: 0.4 4.85 (0.81/CN-)

'Aqueous (HzO) solution of KCN at pH 7 11. *Aqueous (D20) solution of KCN at pD 7 11. Solution of KCN in pyridine/water (3:l). Not measured.

e Solution of KCN in aqueous pyridine solutions of hemin chloride as described under ' ' E x ~ r ~ m e n ~ Proce-

'Solutions of KCN in water a t pH < 8.

h Solutions of KaFe(CN)e in H20. ' Solutions of I&Fe(CN)G in HzO.

dures."

Solutions of KCN in DzO at pD < 8 (DCN) = 100 mM.

TABLE 11 Isotope shifts in C-N stretch frequency for hemoprotein, hemin, and other cyanides

All frequency values are in cm". E x ~ r i m e n ~ conditions are as in the text, The accuracy of d e ~ r m ~ a t i o n s is the same as given in Table I.

shift "N shift Cyanide species Medium UIw14N UWl4N

Observed Calculated" vl*lsa

Observed Calculated"

CN- HzO or D20b 2079 2037 42 43.5 2048 31 32.2 CN- Pyridine/H*O 2075 2033 42 43.4 2044 31 32.2 CN- Pyridine/HzO 2082 2048 34 43.6 2050 32 32.3

HCN Hz0 2093 2060 33 43.8 2060 33 32.5 DCN DzO 1887 1878 9 39.5 1862.5 24.5 29.3 Hemin B pyridine, CN Pyridine/HzO 2126 2081 45 44.5 2095 31 33.0 2,4-Diacetyldeuterohemin IX Pyridine/HzO 2127 2082.5 44.5 44.5 2096 31 33.0

plus hemin

MethemogIobin A CN (human) Hz0 Metchlorocruorin CN (sea Hz0

pyridine, CN 2122 2077.5 44.4 44.5 2092 30 32.9 2125 2079 46 44.5 2093 32 32.9

Metmyoglobin CN (horse) Hz0 2126 2080 46 44.5 2095 31 33.0 Oxidized peroxidase CN (horse- Hz0 2131 2085 47 44.6 2099 33 33.1

Reduced peroxidase CN (horse- Hz0 2024 1984 41 42.4 1995 29 31.4

worm)

radish)

radish) Computed on the basis of a simple diatomic vibrator (Ref. 3). Solutions of KCN in either HzO or DzO gave the same '% and 16N isotopic shifts.

in absorbance in either the infrared or the visible bands was observed beyond the point in the addition of cyanide where the total cyanide concentration became 18 mM, the concen- tration equivalent to metMb; the 2093-cm" band due to free HCN appeared only when the total cyanide concentration exceeds 18 mM.

To obtain as favorable a signal-to-noise ratio (without any smoothing manipulations of the data), as is shown in Fig. lA, required the averaging of nine single scans even though a protein concentration as high as 10 mM was used. The 2126- cm" band for cyanide bound to metMb is only about one- tenth the intensity of the major C - 0 stretch band for MbCO (13) or HbACO (14). On this basis, C-0 stretch spectra of comparable quality can be expected from a hemeprotein car- bonyl solution at one-tenth the concentration of that required to obtain C-N stretch spectra for a hemeprotein cyanide.

The asymmetry of the metMbCN band at 2126 cm" is apparent in the spectrum of Fig. 3A for a solution from the addition of 18 mM metMb and 30 mM total cyanide. Fig. 3B illustrates the lack of success of an attempt to fit the observed

2126-cm" band with a single theoretical band of Gaussian shape (13). As shown in Fig. 3C, the observed absorption band can be satisfac~rily deconvolut~ into two Gaussian shaped bands.

Infrared spectra for the other oxidized hemeproteins yihded similar C-N stretch band parameters (Tables I1 and V, Fig. 4). MetHbA had the lowest VCN value (2122 cm") and HRP(III)CN2 the highest (2131 cm"). MetHbA exhibited the narrowest band ( A V ~ = 7.0 cm") and HRP(III)CN, with a value of 12.0 cm", the widest. The band intensity is much greater for H R P ( 1 I I ~ C ~ than for all the other iron(II1) pro- teins. Replacement of HzO by DzO did not affect the U C N for the other iron(II1) hemeproteins significantly. Isotope effects due to 13C and 16N were similar to those found for hemin cyanides.

Attempts to prepare Fe(I1) cyanides for Mb, HbA, chloro- cruorin and horseradish peroxidase were successful only in

The abbrev~tions used are: HRP(II)CN, the cyanide complex of reduced horseradish peroxidase; HRP(III)CN, the cyanide complex of oxidized horseradish peroxidase.

Infrared Spectra of Cyanide ~o~~ to H e ~ e p r ~ ~ e i ~ 3521

TABLE 111 Infrared, visible-Soret, an$ proton magnetic resonance spectral data for 2,4-substituted deuterohemin I X cyanides

Spectra recorded at 20 "C for DnO/pyridine-da solutions about 45 mM in hemin prepared as described in text. Ring substituent PMR chemical shifts 2,4- Axial Visible-Soret

Substituent ligands CH, aCHl BCHl Methine 2.4-R maxima UCN

H Pyridine-F+CN

H NC-Fe--CN

H --C=SCHz Pyridine-FAN

HI -C=SCHg N C - F A N

0 -CCHs Pyridine-Fe-CN

0 "cCW8 NC-F+-CN

em" 2124.5

2112.0

2125.5

2112.5

2127.5

2114.0

nm 529.5 407.0

539.5 418.5

542.0 417.5

554.0 433.0

551.5 425.5

569.0 443.0

-20.53 -17.66 -16.62 -14.85 -18.47 -15.97 -14.51 -12.51

-19.95 -19.43 -15.45 -14.12

-17.63 -17.13 -13.24 -11.69

-26.25 -19.98 -12.33 -4.88

-20.13 -19.68 -9.69 -3.88

-8.66 -7.34

-7.89 -6.81

-8.00 -6.76

-7.41 -7.12

-9.09 -6.88

-7.88 -6.05

PPm -0.70 -0.53

-0.76 -0.60

-0.65

-0.66

-1.31 -0.87

-1.61 -1.28

-4.84 -3.75 -3.29 -2.91 -3.57 -1.28 -0.76 -0.51

-5.10 -3.21 -2.98 -2.56

-3.42 -0.59 -0.12 +0.70

-1.76 -0.69 -0.33

E

i-0.18 +2.87 +6.46

+18.51 +19.08

+I553

-13.20" -12.31" +3.02' +3.49'

-12.08' -11.20" +1.61' +2.196

-1.98 -1.92

-2.06 -2.02

a C H 4 H Z . * CR=€&. Not assigned.

TABLE IV Infrared parameters for the C-N stretch band for "C"N- bound to

substituted deutenohernins IX All spectra were recorded at 25 "C in aqueous pyridine solutions

prepared as described in the text.

Substituents 2,4-

linands Axial

W N AV,' 8 - cm" C 0.5 em" f 1.0 m M ' em"

Hydrogen Pyridine-Fe-CN 2124.5 16.5 0.03 Vinyl Pyridine-Fe-CN 2126 15.0 0.04 Acetyl Pyridine-FeCN 2127 15.5 0.04 Hydrogen NC-Fe--CN 2112.0 11.0 0.10 Vinyl N C - F A N 2112.5 11.0 0.12 Acetyl N C F d N 2114.0 11.0 0.12 "The m e ~ ~ e m e n ~ of AY, values for the monocyanides were

carried out on highly symmetrical bands. However, the &cyanide bands were somewhat asymmetric.

Extinction at band maximum per cyanide ligand.

the case of horseradish peroxidase. Evidence of cyanide bind- ing to heme iron(I1) was sought in terms of visible-Soret and infrared spectra under a variety of conditions of pH up to 9.4 and cyanide up to 100 mM. Keilin and Hartree (11) reported that a stable deoxyMbCN was obtained if M e t ~ C N was reduced with sodium dithionite in the presence of 1 M cyanide at pH 9.3. However, under the conditions used here, no evidence of cyanide binding was obtained for the deoxy species of HbA, IMb, or chlorocruorin. On the other hand, at pH > 8.0, a C-N stretch band of cyanide bound to HRP(I1) was observed and this band is at a much lower frequency, wider,

and more intense than the HRP(II1)CN band (Tables I1 and V). Confirmation of the oxidation state was obtained from the visible-Soret spectrum which was the same as the reported spectrum for HRP(1I)CN (11). Additional confirmation was obtained upon exposure of the solution to CU whereupon the C-N stretch band near 2030 cm" disappeared completely and bands in the C-0 stretch region appeared. The carbonyl spectrum contained an asymmetric band (AvH = 16 cm") at 1934 cm" with a shoulder at 1928 cm-l. Also, about 15% of the total intensity was in two bands with maxima near 1957 and 1910 cm". An identical multiple banded C - 0 stretch spectrum was obtained in the absence of cyanide. The spec- trum resembles the C-0 spectrum of alkaline type for horse- radish peroxidase carbonyl at pH 11 reported by Barlow et al. (16). These data show that the cyanide ligand is easily re- pIaced from HRP(I1)CN by CO.

The C-N stretch band for HRP(I1)CN is shifted to higher frequency by about 8 cm" upon exchange of D20 for H20. HRP(1I)CN is the only hemeprotein where D20 exchange had such a marked effect. The asymmetric HRP(I1)CN band can be deconvoluted into symmetric bands of Gaussian shape as shown in Fig. 5. The two calculated bands obtained from the band observed in H,O are both at a 8 cm" lower frequency than the corresponding bands obtained in D20. The band widths of the calculated bands (-23 cm") are the same in D20 or H20, but the relative band intensities differ. Both the shape and the intensity of the HRP(I1)-CN stretch bands were found to vary among different samples of protein ob- tained commercially. Typical results are given in Fig. 5 and

3522

W 0 z a m

m a

c 8

Infrared Spectra of Cyanide Bound to Hemeproteins

r - 1

I J 2140 2120 2100

8 metMb- CN*

L I

2100 2080 x)60

WAVENUMBER (cm") FIG. 1. Effects of isotope substitution on infrared spectra

for cyanide bound to horse metmyoglobin. The sample cell contained 10 mM metMb and (A) 42 mM total cyanide as 12C1'N, ( B ) 24 mM total cyanide a s W 5 N , and ( C ) 39 mM total cyanide as I3Cl4N in 10 mM sodium phosphate buffer, pH 7.4, at 23 "C. The reference cell contained water.

Table V. For the several samples we examined, UCN values in HzO are between 2025 and 2034 cm", Avw values are between 33 and 36 cm", and t values are between 0.42 and 0.47 mM" cm". The effects of DzO exchange and isotopic substitution in cyanide reported here were all determined on a single sample of the protein. It should be noted that horseradish peroxidase preparations can contain different relative amounts of isoenzymes; the supplier of the horseradish per- oxidase used here stated that two basic and no acidic isoen- zymes are present in their preparations.

DISCUSSION

Measurement of C-N Stretch Bands for Cyanide Bound to Hemeproteins-The measurement of C-N stretch bands at useful signal-to-noise levels is limited by low band intensities which may require concentrations above 1 mM and by the

TABLE V Infrared parameters for the C-N stretch band for "C1'N- bound to

hememoteins in H20 or D20 buffers

Protein Oxidation Hz0 or state D,O WCN Au," t

Hemoglobin A (human)

Hemoglobin A (human)

Hemoglobin (lamprey)

Chlorocruorin (sea worm)

Myoglobin (horse)

Myoglobin (horse)

Peroxidase (horseradish)

Peroxidase (horseradish)

Peroxidase (horseradish)

Peroxidase (horseradish)

em" cm" rnM" em"

Fe3+ H20 2122 f 0.3 7.0 f 0.6 0.07

Fe3+ D20 2122 f 0.3 7.0 f 0.6 0.07

Fe3+ H20 2128 & 0.5 9.0 f 1.0 0.06

Fe3+ H20 2125 f 0.5 10.0 f 1.0 0.05

Fe3+ H20 2126 f 0.3 10.0 f 0.7 0.07

Fe3+ D20 2125 f 0.3 10.0 f 0.7 0.07

Fe3+ H20 2131 f 0.5 12.0 f 1.0 0.24

Fe3+ D20 2131 f 0.5 12.0 f 1.0 0.24

Fez+ H20 2029 f 0.8 36.0 f 2.0 0.42

Fez+ D20 2037 & 0.8 28.0 f 2.0 0.55

"Band widths are for bands as observed. In several cases, the observed band has been deconvoluted into two bands (Figs. 3-5).

0.6 h] O'Oo6

- 0.004 '5

'u 0

(u e 0

4

0.002 Q

IO 20 CYANIDE (mM)

FIG. 2. Changes in visible and infrared band intensities upon titration of horse metmyoglobin with cyanide. A solution of 18 mM metMb in 20 mM sodium phosphate, pH 7.4, at 23 "C was treated with increasing amounts of a neutralized aqueous solution of KCN. After each addition of cyanide, the changes in absorbance between 540 and 630 nm and at 2126 cm" were measured. For the infrared measurements, the reference cell contained 18 mM metMb in the same buffer but no cyanide.

need to keep infrared cell pathlengths short (about 0.05 mm or less) for aqueous media due to the high absorption of energy by water (3). The signal-to-noise ratio can be improved significantly by use of DzO as a medium because, near 2100 cm", absorbance by DzO is much weaker than by H20. For these measurements, where a range of only about 100 cm-I or less need be covered and precise difference spectroscopy is critical, the Fourier transform infrared spectrometers that are currently available commercially do not provide any advan- tage in terms of spectral quality compared with the spectra obtained here with a high quality dispersive instrument.

Bands due to metal bound cyanide can be distinguished from bands due to free cyanide (CN- or HCN) by use of DzO and/or I3Cl4N-. Since exchange of DzO for HzO shifts VCN

Infrared Spectra of Cyanide Bound to Hemeproteins 3523

I , I

t 4 1 0.001 1

I I 2 I40 2120 2 100 WAVENUMBER (cm")

FIG. 3. Deconvolution of the C-N stretch band of horse met- myoglobin cyanide. A , infrared spectrum from 16 accumulated and averaged single scans for 18 mM metMb and 30 mM total cyanide in 20 mM sodium phosphate, pH 7.4, at 23 "C. The bands at 2126 and 2093 cm" are due to hemin-bound CN- and free HCN, respectively. B, an attempt to fit the observed band (a) from A to only one computed band ( b ) of Gaussian shape. Tracing c represents spectrum a minus curue b and shows residual absorption at 2132 cm". C, deconvolution of observed band ( a ) into two curves (b and c ) whose sum subtracted from Q gives d which lies closely on the base-line in the region of the bound CN- band. Band parameters for computed bands: b, u = 2125.7 cm", Auu = 8.4 cm", and c = 0.050 mM-' cm-l; and c, u = 2132.2 cm", AU, = 5.0 cm", and c = 0.010 mM" cm".

from 2093 cm" for HCN to 1887 cm" for DCN (Table I), such a shift indicates an absorption a t 2093 cm-I in water solutions is due to HCN. A metal cyanide band present in the region of the HCN band is shifted little, if any, by DzO (Tables I1 and V). The use of 13C14N- in place of '*C14N- shifts uCN for HCN 33 cm" compared with a calculated shift of 43.8 cm-l,

w V Z a m LT 0 cn a a

2150 21 30 21 IO 2090

F"-- Met Hb-CN (DzO)

21 30 21 IO 2090 2070 WAVENUMBER (cm")

FIG. 4. Infrared spectrum for cyanide bound to oxidized horseradish peroxidase and metHbA. A, oxidized horseradish peroxidase (2 mM) and 19.2 mM total cyanide in 0.1 M sodium phosphate buffer, pH 8.0. The reference cell contained water. B, MetHbA (12.8 mM) and 50 mM total cyanide in 10 mM sodium phosphate, pD 7.4, in D20. The reference cell contained 12.8 mM metHb in D20 without cyanide. Upper tracing, the spectrum obtained experimentally from 36 accumulated and averaged single scans. A weak band near 2090 cm" is from HCN due to incomplete D20 exchange. The smooth curues represent deconvolution of the 2122 cm" band into two Gaussian curves. Lower tracing, the difference spectrum from the observed spectrum minus the sum of the two computed curves. Band parameters for the computed curves are u = 2127.7 cm", Au, = 7.0 cm", c = 0.015 m"' cm" and u = 2122.2 cm", Au, = 5.9 cm", E = 0.067 mM" cm".

whereas for iron cyanides the observed shift is very near (& 2.5 cm") the calculated value (Table 11). These cyanide infrared band characteristics have proven critically useful for the identification of cyanide bound to Fe and Cu sites in cytochrome c oxidase (15), as will be reported in detail else- where.

Variations in C-N Stretch Band Parametrs for Hemeprotein and Hemin Cyanides-A difference in VCN of 102 cm" results from a change in oxidation state of the peroxidase in water (Table V). UCN values for the oxidized hemeproteins vary only 9 cm" due to differences in protein structure. Changes in porphyrin structure causes shifts in ucN for monopyridine monocyano hemins from 2124.5 to 2127 cm" and dicyanides from 2112 to 2114 cm" (Table IV). Replacement of pyridine of a monopyridine monocyanohemin with cyanide decreases UCN by -13 cm". The 13C and 15N isotope shifts for iron bound

LLJ 0 2: a

0 #

4 a

~~

2060 2020 1980 WAVENUMBER (cm")

FIG. 5. Effects of DBO~H~O exchange in the C-N stretching bands of HRP(I1)-CN. A , reduced horseradish peroxidase (1.30 mM) and 40 mM total cyanide in 0.1 M sodium phosphate buffer, pH 8.0, in H20. The reference cell contained the protein solution without cyanide. The e x ~ r ~ m e n ~ I i y obtained curve (upper tracing) is decon- voluted into two Gaussian curves (smooth lines). The lower tracing is the difference spectrum between the observed spectrum minus the sum of the two computed curves. Band parameters for these curves are v = 2028 em", Am., = 23 cm", c = 0.41 mM" em-' and u = 2007 cm", buu = 23 cm", t = 0.20 mM" cm-'. B, reduced horseradish peroxidase (0.8 mM) and 40 mM total cyanide in 0.1 M sodium phosphate buffer, pD 8.0, in D20. The observed spectrum, deconvo- lution, and difference spectrum were obtained as in A. Band param- eters for the computed curves are v = 2036 cm", A% = 24 cm-I, t = 0.51 mM" em" and v =I 2013 em", AUW = 22 cm", t = 0.10 mM" cm-l. The absorbance increase in the higher wavenumber region in spectrum A is due to HCN free in solution and the increase in the lower wavenumber region in spectrum B is due to DCN.

cyanides are near the calculated values based on the simple diatomic model (3), -45 cm" for 13C and -33 cm" for I5N (Table 11). The substitution of D20 for H20 increases UCN by- as much as 8 cm" for HRP(I1)CN but no significant effect on uCN is observed for the other hemeproteins (Table V). Band widths (hulk) for the oxidized hemeproteins vary from 7.0 cm" (metHbACN) to 12.0 cm" (HRP(II1)CN) (Table V).

HRP(I1)CN exhibits the widest (AuH = 36 cm") and the most intense hemeprotein band. Among the hemin cyanides, changes in 2,4-substituents have little effect upon band widths and intensities (Table IV). However, bands for the dicyanides (Av, = 11 cm-') are uniformly narrower than are bands for monopyridine monocyanides (AuH = -16 cm").

Reasons for V u r i a t ~ ~ in C-N Band Freqwney- The bond- ing of CO or O2 to a metal ion results in a C - 0 or 0-0 stretch frequency of the metal bound ligand that is always greatly reduced from the gaseous value (14, 17-19). In contrast, uCN

may either increase or decrease when CN- binds to a metal ion (20). Thus, ferricyanide (2116 cm") is increased 37 cm" and ferrocyanide (2037 cm") is decreased 42 cm" from the anion value of 2079 cm-l. Similarly, HRP(II1)CN at 2131 cm" is increased and HRP(I1)CN at 2025 cm" is decreased. Protonation of CN- in water to form HCN causes a shift in UCN from 2079 cm" to 2093 cm". This shift due to protonation has been explained as due to the proton accepting electrons as a a-acceptor from carbon, thereby increasing both the C- N bond order and the frequency of the C-N vibration (20): The shift to lower frequency by the Fez+ of ferrocyanide and HRP(I1)CN is consistent with Fez+ being a less effective a- acceptor than H+ and/or the low-spin Fez+ being an effective dx-donor. The opposite shift in UCN by the Fe3+ compounds is explained as due to Fe3+ being more effective than is H+ as a a-acceptor. Also, Fe3+ i s less effective than Fez+ as a d?r-donor.

The smaller shifts in uCN that occur with the exchange of ligands, while maintaining the same (Fe3+) oxidation state, may also be explained in terms of a-acceptor and dx-donor differences. The hemin dicyanides, which represent the re- placement of four in-plane cyanides of ferricyanide by four porphyrin nitrogens, exhibit VCN values from 2112 to 2114 cm" which are only sightly below the ferricyanide value of 2116 cm" (Tables I and IV). Thus, the overall effect of the four cyano ligands is nearly the same as the porphyrin ligands. Changes in 2,4-substituents that decrease porphyrin nitrogen basicity in the order hydrogen > vinyl > acetyl (21) results in only small increases in UCN (Table IV). These shifts, due to the effect of changes in porphyrin on an axial ligand known as a cis- or H-effect (23) are in the direction expected in that a decrease in porphyrin nitrogen basicity results in Fe3+ being a more effective a-acceptor and a less effective dr-donor, which will increase VCN. Since only small shifts in UCN result from H-effects in the hemins, the vCs of 2125 cm" found for oxidized chlorocruorin CN which contains 2-formyl-4-vi- nyldeuterohemin IX is expected to be near the VCN values for hemeproteins that contain 2,4-divinyldeuterohemin IX (hemin B): lamprey metHbCN (2128 cm-l), m e t ~ C N (2126 cm"), and metHbACN (2122 cm") (Table V). The replace- ment of a 2-vinyl with a 2-formyl gives a less basic porphyrin with a basicity comparable to the 2,4-diacetyl derivative (21, 24). The C-0 stretch frequencies of heme carbonyls are much more sensitive to H-effects than are C-N stretch frequencies. For example, the same changes in 2,4-substituent that result in a 9 cm" shift in uco for hemepyridinecarbonyls (17) result in only a 2-cm-' shift in UCN of the respective m o n o p ~ i ~ n e monocyanides.

The proximal or trans effect due to replacement of one of

The very large lowering of UCN for cyanide when in nonalkaline DzO (ie. a shift from 2079 cm" to 1887 cm") (TabIe I) is not due solely to the bonding of D' to CN- to form DCN. At least some of the shift is reasonably ascribed to association of DCN molecules. For example, whereas the monomeric DCN in an argon matrix at 20 K has a vCN of 1924.5 em", the multimer value is 1886 em", near the solid value of 1885 cm". In contrast, the monomeric HCN value is 2088.8 cm" with multimer and solid values of 2110.1 and 2099 cm", respectively (22).

Infrared Spectra of Cyanide Bound to Hemeproteins 3525

the cyanides of a hemin dicyanide by pyridine results in a shift to higher frequency of 13 cm" (Tables 111 and IV). Simil'arly, uCN values for the synthetic tetraphenylporphine iron(II1) complexes in KBr were recently reported as 2120 cm" for the dicyanide (25) and 2130 cm" for the pyridine cyanide (26). Replacement of trans-cyanide by pyridine ren- ders Fe3+ a less effective d*-donor and a more effective u- acceptor to cause a shift to higher frequency. Both visible and Soret maxima are markedly blue-shifted in pyridine-Fe3+-CN compared with NC-Fe3+-CN (Table 111). A blue shift is con- sistent with an increased ability of the Fe3+ ion to accept electron density from the porphyrin nitrogens (27). 'H NMR spectra also reflect a stronger interaction between the low- spin paramagnetic Fe3+ and the porphyrin nitrogens for pyr- idine-Fe3+-CN than for NC-Fe3'-CN; the paramagnetic shifts of resonances for porphyrin ring substituents are much greater for the monocyanide (Table 111) (28). The crystal structures reported by Schiedt and co-workers (25, 26) for analogous tetraphenylporphine derivatives revealed stereochemical dif- ferences between pyridine-Fe3+-CN and NC-Fe3+-CN species. For example, the Fe atom is out of the porphyrin plane towards the CN- by 0.03 A in the monocyanide but in-plane in the dicyanide. The FeCN bonds were linear in both com- pounds but the Fe-C bond was off 3.6" from the heme normal in the monopyridine but only off 1.6" in the dicyanide. The distal atom of the cyanide (presumably nitrogen) was hydro- gen bonded to water in the monocyanide. An interaction between a distal atom and a potassium ion was found in the dicyanide crystal. We assume there is a close correspondence at the cyanide binding sites between the Scheidt crystal structures and the solution structures for our substituted deuterohemins. Furthermore, the close agreement between uCN values for hemin B monopyridine monocyanide and those for the oxidized hemeprotein cyanides suggests the mono- cyanide represents a close model of the hemin B cyanide within the hemeproteins. The wavelengths of visible-Soret absorption maxima of the monocyanide also agree much more closely with the hemeproteins than does the dicyanide. For example, band maxima are found at 540 and 422 nm for metMbCN (29) and 540 and 421 nm for metHbACN, com- pared with 542.0 and 417.5 nm for hemin B monopyridine monocyanide and 554.0 and 433 nm for the dicyanide.

Characteristic Variations in C-N Band Width and Znten- sity-Width (Amh) and intensity (e) values vary significantly among the hemeproteins studied (Table V). The band width reflects the diversity (lack of uniformity) of the intimate environment about the C-N vibrators (4, 13). A comparison of AuH values for the hemin B monocyanide (15.0 cm") with the same hemin in metHbACN (7.0 cm") provides a striking example of environmental effects provided by aqueous pyri- dine compared with the metHbA protein (Tables IV and V). The widths for cyanide bound to the hemin and for free cyanide anion (16.5 cm-') are similar (Table I), which reflects a similar diversity in the interactions between C-N and sol- vating molecules. In contrast, interactions of the cyanide ligand of metHbACN with amino acid residues from the globin, and possibly with a water molecule as well, are much less diverse than are the interactions of the cyanide of the hemin cyanide with aqueous pyridine. The environments pro- vided by the other proteins result in broader bands than those for metHbACN; e.g. Aulh for metMbCN is 10 cm", HRP(II1)CN 12.0 cm", and HRP(I1)CN 36 cm". The widths of major carbonyl bands also increases in the order HbACO < MbCO < HRP(1I)CO. Thus, both cyanide and carbonyl stretch bands reveal the same order of increasing diversity in solvating environment. Metal oxidation state can also mark-

edly affect widths. For the hexacyanides, the iron(II1) complex band is much narrower than the band for the iron(I1) complex (Table I). This finding suggests that the distribution of three negative charges over six CN- ligands is easier to achieve in a highly symmetrical fashion than is the distribution of four negative charges. With horseradish peroxidase, the higher oxidation state band is also much narrower. The CN ligand environment thus appears much more diverse for HRP(1I)CN than for HRP(II1)CN. Here, of course, the presence of two conformers must also be considered (Fig. 5).

Significant differences in intensity occur among hemepro- tein cyanides (Table V). Both the extinction and the int,e- grated area (B values) of C-N stretch bands serve as useful characteristics of these cyanides. Wide variations in intensity are a characteristic of metal cyanides generally; values may differ by several orders of magnitude (20). However, little is known at present about the relationship between the intensity of a C-N stretch band and the structure of the metal complex or the nature of the medium.

HCN versus CN- Binding-The substitution of 13C for 12C and of 15N for 14N causes shifts in ucN that are very near those expected from simple reduced mass calculations in the cases of cyanide anion, the hemin cyanides, and the hemeprotein cyanides (Table TI). However, with HCN in HzO, the I5N isotope shift agrees with the calculated value but the 13C shift is lower than the calculated value by 11 cm". The accepted usual mode of bonding between cyanide and a metal ion involves formation of a metal-carbon bond as in Structure I. However, a few metal complexes are now considered to con- tain HCN bound to the metal via nitrogen in the manner of bonding between a metal ion and a nitrile (M-NCR) (31) as in Structure 11. A recent review of such HCN-metal complexes included no iron complex (32). In confirmation of their struc- tures, appropriate C-H bands in the infrared and C-H reso- nances in NMR spectra are observed for these nitrile-like HCN-metal complexes; however, no crystal structures have been reported. The C-N stretch frequencies found are some- what higher than the HCN values; many are near those found here for hemin and hemeprotein cyanides. Therefore, a struc- ture as in Structure I1 can not be excluded for hemins or hemeproteins on the basis of a comparison of uCN values for hemin-bound cyanide with values for the small HCN-metal complexes. Nevertheless, the presence of nitrile-like HCN bonding to iron in the hemeproteins studied here is unlikely for several reasons. As discussed above, if HCN were in fact found in an end-on nitrile-like fashion, a less than calculated 13C shift is very probable (Table II), whereas the calculated shift is found for all hemeprotein and hemin cyanides. Fur- thermore a characteristic feature of the HCN-metal com- pounds is their incompatibility with water (32). Since the vCN

for DCN in D20 is 206 cm" lower than the value for HCN in Hz02 (Table II), if HCN is in fact bound to a hemeprotein as in Structure 11, then a large decrease in YCN may be expected

F e - C r N STRUCTURE I

Fe . . . N&-H STRUCTURE I1

H

C Fe . . . 111

N STRUCTURE I11

I

3526 Infrared Spectra of Cyanide Bound to Hemeproteins

upon the exchange of DCN for HCN in a hemeprotein cyanide solution. No such decrease in UCN is observed (Table V). The cyanide infrared spectra are also inconsistent with a second type of HCN bonding, namely, side-on bonding (Structure 111) with the CN bond parallel to the heme plane in analogy to olefinic-metal bonding. All known compounds of such bonding with nitriles exhibit far lower UCN values than we have found for hemeprotein cyanides (31, 32).

Stereochemistry of Cyanide Bonding: Bending Versus Tilt- ing-Crystal structures for several hemeprotein cyanides have been reported, i.e. metMbCN (33), metlamprey CN (34), horse metHbCN (35), meterythrocruorin CN (36) and cytochrome c peroxidase CN (37). It is clear in each of the protein structures that the CN bond is decidedly off the normal to the heme plane, i.e. the Fe-C-N bonds appear to be bent. The steric constraints provided by amino acid residues of the protein appear to force nonlinearity on the Fe-C-N bonds in the manner also noted in hemeprotein carbonyl structures (38-40). Since, in the absence of steric constraints, linear Fe- C-N bonding is favored energetically over a bent configura- tion, it was suggested that the bending of the Fe-C-N bonds was only apparent and did not occur; rather, the Fe-C-N bonds remained linear while the porphyrin ring became dis- torted from planarity, a phenomenon described as tilting (35, 37). In fact, steric pressure on the distal N of the cyanide ligand will undoubtedly result in both bending and tilting (Fig. 6). The key question then is what are the relative energies of "bending" and "tilting"; the extent to which bend- ing versus tilting occurs will depend upon their respective energy requirements. The structural results obtained by Scheidt and co-workers (26) for the tetraphenylporphine iron(II1) cyanides led them to suggest a preference for tilting over bending. However, protein crystallography cannot be expected to achieve sufficient resolution to provide detailed evidence of the relative contributions of bending and tilting. Infrared spectroscopy can thus represent a uniquely useful approach.

Our infrared data provide evidence that tilting may require less energy than bending since there is no apparent infrared evidence of a qualitative difference in FeCN bonding between

N I

I / I /

BENDING TILTING FIG. 6. Schematic representations of bending and tilting

due to steric pressure upon the cyanide ligand from distal amino acid residues of the protein. The vertical dashed line represents the average porphyrin plane. The arrows indicate the direction of steric pressure. In the absence of steric pressure, linear Fe-C-N bonding is favored energetically. In pure bending the only change due to steric pressure is a decrease in the Fe-C-N angle from 180". In pure tilting the Fe-C-N bonding remains linear but the C-N bond is off-axis to the average porphyrin plane due to distortions in the four pyrrole portions of the prophyrin ring. Steric pressure will in fact result in both bending and tilting with the extent of each dependent upon their relative energy requirements.

the hemins and the hemeproteins. The tilting phenomenon per se is unlikely to alter significantly either the stereochem- istry between Fe, C, and N or the bonding between these atoms. On the other hand, bending is expected to change the bonding between Fe and C as well as between C and N. For hemeprotein carbonyls the observed deviations of the C-0 bond from the heme normal increases in the following or- der:HbA b-subunits (13"), HbA a-subunits (14"), and MbCO (24") (39, 40). This is also the order of decreasing uco for major bands: HbA @-subunit (1951.8 cm"), HbA a-subunit (1950.4 cm"), and MbCO (1944 and 1933 cm") (13,41). The order of increasing UCN of metHbACN < horse metMbCN < HRP(II1)CN may well represent the order of increasing ap- parent bending of the Fe-C-N bonds. Carbonyl and cyanide hemeproteins are similar in several respects. Both CO and CN- are diatomic ligands that energetically favor linear iron- ligand bonding (Fe-C-0 or Fe-C-N). Both Fe2+C0 and Fe3+CN- have formally neutral (uncharged) ligands. The two ligands can therefore be expected to experience similar steric pressures from a given protein structure. The fact that the stretch frequencies shift in opposite directions with increasing steric pressure (uco decreases, uCN increases) should not be considered evidence that the steric effects are not the same, since H-bonding or attachment of another group to the oxygen atom of a carbonyl ligand decreases uco (42) whereas bonding of an atom or group to the nitrogen of a cyanide ligand results in increased uCN (43-45).

A consideration of I3C isotope shifts provides another ap- proach to the bending versus tilting problem. Although ad- mittedly only on the borderline of being significant, the dif- ferences in I3C isotope shift relative to calculated values increase in the order metHbACN < metMbCN < HRP(II1)CN (Table 11). The I3C isotope shifts are likely to be influenced more by bending than by tilting since tilting is expected to influence the bonding between Fe, C, and N atoms less than bending, as discussed above. Therefore, if the ob- served variations in the difference between observed and calculated 13C shifts with changes in protein structure are in fact real, then, in the future, very accurate measurements of such differences might provide a uniquely useful approach to the evaluation of Fe-C-N bending in hemeprotein cyanides.

Multiple Conformers-The ligand binding site structures of hemeproteins may involve multiple protein conformers in which there is hydrogen bonding between the ligand and protein amino acid residues or solvent (H20) molecules. In- frared spectra can be expected to provide data relevant to the presence of both conformers and hydrogen bonding. Conform- ers present in myoglobin and hemoglobin carbonyls (13, 46, 47) have been identified and directly measured only by CO infrared spectra where four infrared bands ranging in fre- quency from near 1930 cm" to 1970 cm-I are found. The four discrete infrared bands are due to four conformers of signifi- cantly different structures which can interconvert rapidly. Here, infrared spectroscopy has an advantage over 13C NMR spectroscopy in that the NMR method, due to its slower time scale, only reveals a single 13C resonance representing the weighted average for the four interconverting conformers (13). Two different infrared bands are seen for azide bound to sperm whale metMb and to human metHbA (5, 27). The two azide infrared bands also represent different protein conform- ers, one with a high-spin hemin azide and the other a low- spin hemin azide. Deconvolution of high signal-to-noise C-N infrared spectra indicate multiple conformers can be present also in hemeprotein cyanides. Two bands are present in spectra for horse metMbCN (Fig. 3), metHbACN (Fig. 41, and HRP(I1)CN (Fig. 5). The HRP(II1)CN spectrum can also be

Infrared Spectra of Cyanide Bound to Hemeproteins 3527

deconvoluted into at least two bands. In the case of the horseradish peroxidase derivatives, the ~ s s i b i ~ i t y of the bands being due to multiple isozymes exists-the supplier states only two basic isozymes are present. However, the two bands obtained upon deconvolution of HRP(I1)CN spectra in HzO and in DzO can not be explained as due entirely to two isozymes because the relative intensities of the two bands vary wtih solvent-H20 or DZO (Fig. 5).

Hydrogen Bonding-The differences between the DzO and HzO spectra of HRP(1I)CN are likely due to the effects of hydrogen bonding between the bound cyanide ligand and an amino acid residue, presumably histidine. However, effects on protein structure due to the exchange of protons for deuterons generally throughout the protein can not be excluded unequiv- ocally. The hydrogen bonding is envisioned as being to the terminal nitrogen of bound cyanide anion as in Structure Ia which is distinctly different from HCN bonding in the manner of a nitrile as in Structure 11. Hydrogen bonding to cyanide as in Structure Ia has been proposed for cyanide anion in water (48), the aggregation of HCN (49), the crystal structure of tetraphenylporphine iron(II1) monopyridine monocyanide (26), and the hemeprotein cyanide structures (33-37). 'H NMR evidence for such hydrogen bonding between a distal histidine and bound cyanide has been reported for metMbCN (50,51). The iack of detectable effects on infrared bands due to the exchange of H20 by D20 in the cases of metHbACN, m e t ~ C N , and HRP(II1)CN (Table V) can result from weaker H-bonding in the iron(II1) cyanides than in the iron(f1) cyanide. Cyanide can form stronger H-bonds with protein residues when bound to Fe2+ than when bound to Fe" of hemeproteins because the Fe2+CN will bear an unneutral- ized negative charge. On the other hand, Fe3+CN- will be formally uncharged overall.The electrostatic interaction be- tween negative cyanide anion and positive hemin iron is expected to make a major contribution to the usually high affinity of an oxidized hemeprotein for cyanide. The strength of the Fe-C bond per se is not sufficient to have much effect on the shifts in vCN upon replacement of "C by 13C. In other words, the Fe-C bond is so weak that the C-N vibrator behaves like the free anion in terms of isotope shifts. In contrast, formation of the strong H-C bond in HCN markedly dimin- ishes the shift due to this isotope substitution compared to shift for the free anion (Table 11). The relatively long Fe-C bonds found in the crystal structure of a hemin dicyanide (25) also demonstrate the presence of weak Fe-C bonds in a hemin cyanide. Therefore, hydrogen bonding between distal residues and cyanide ligand as in Structure Ia, even in the iron(II1) cyanides, can contribute importantly to the affinity of the protein for cyanide. Furthermore, in proteins where more than one C-N stretch band are seen in accord with the presence of multiple conformers at the ligand binding site, the H-bonding interactions may well differ among conform- ers. it now seems apparent that several factors influence cyanide affinity; these include steric accommodation and charge neutralization via interactions between distal protein residues and cyanide ligand as well as formation of an iron- carbon bond. Clearly, the equilibrium constant for cyanide binding to a hemoprotein is not necessarily directly related to the strength of the Fe-C bond formed.

The shift in the relative intensity of the two bands for HRP(I1)CN upon replacement of HzO with DzO (presumably due to a change in the relative stability of two conformers) suggests H-bonding stabilizes at least one conformer. Horse-

Fe-C=N. . . H+-R

STRUCTURE Ia

radish peroxidase apparently has a proton donor (e.g. histi- dine) at the active site that is important for catalysis (16,19, 52, 53). The proton donor could also participate in cyanide binding. The cyanide infrared data reported in this paper give clear evidence of a ligand site in horseradish peroxidase that is distinctly different from the sites found in the other oxi- dized hemeproteins. The other protein sites exhibit charac- teristic differences in their C-N stretch bands, but the differ- ences between themselves are less marked than with horse- radish peroxidase. HRP(1II)CN is strikingly different in all C-N stretch parameters-C-N stretch band position, band width, and intensity. Furthermore, of the proteins studied, HRP(1I) is the only reduced species that exhibited a C-N stretch band in the presence of cyanide. Since horseradish peroxidase is an enzyme utilizing hydrogen peroxide as one substrate and the other proteins are in each case an oxygen carrier, the differences in ligand binding site structures ob- served can be expected. Carbon monoxide infrared spectra have also shown the CO binding site of reduced horseradish peroxidase to be distinctly different from the sites in other hemeprotein carbonyls (4, 19, 53). It is now apparent that cyanide and carbonyl infrared spectra can each make uniquely useful contributions to the evaluation of hemeprotein ligand binding site structures.

A c k n o ~ ~ g m n t s - W e thank Professor M. Yoshida, M. Isozaki, and other staff members of the Okayama University Marine Institute, Oku, Okayama, Japan for the identi~cation of the sea worm, Potam- ilk l e p t m k t a , their kind help in the collection of the sea worms, and the friendly hospitality extended by their institute.

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20.

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