Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.

The effects of formylation by chemical modifications (1) of the vinyl groups at positions 2 and 4 of the porphyrin ring on the optical and oxygen binding properties of the ferrous form of human hemoglobin (2) and horse heart myoglobin (3,4) have been studied with respect to the equilibrium (2,3) and kinetic (4) aspects. The formylation of the vinyl groups caused an optical red shift, a decrease in the intrinsic oxygen affinity, and an increase in both combination and dissociation rate constants. Most of these changes are attributed to the withdrawal of the r electron density involved with oxygen binding toward * the periphery of the porphyrin ring. It was also found that the two vinyl groups at positions 2 and 4 of ferrous heme side chains are nonequivalent in their optical, equilibrium and kinetic properties (2)(3)(4).
Heme modification studies of ferric hemoproteins have been reported for various hemoproteins including hemoglobin (5-7), myoglobin (8-ll), horseradish peroxidase (12)(13)(14), and cytochrome c peroxidase (15,16). Makino and Yamazaki (11,12) indicated that the pK values of the dissociation constants of the complexes with various ionic ligands and the pK, values of the transition between the acid and alkaline forms for the reconstituted horseradish peroxidase or sperm whale myoglobin have a close correlation with pK,' values of the metal-free porphyrins.

Hemins
Three kinds of formyl hemin were prepared by a method described elsewhere (1,2). The prep,arations of deutero-and mesohemins were made according to the methods of Chu and Chu (20) and Fischer and Piitzer (15,21), respectively.
A standard technique (15) was used for the incorporation of iron into these porphyrins.
All chemicals of reagent grade were purchased from the following companies and used without further purification: potassium phosphate, potassium cyanide, potassium cyanate, and potassium thiocyanate from Fisher Scientific Co.; potassium fluoride from J T Baker Chemical Co.; and sodium azide from Matheson Coleman and Bell Co.

Acid-Alkaline Transition Measurements
The pK, value of the transition between the acid and alkaline forums of ferric formylmyoglobin was determined spectrophotometrically in both visible and Soret regions and was calculated from the following equation, assuming that n = 1, pK, = n pH ~ log (a~k,~f~~~) where n is the Hill constant. The measurements were made at the wavelengths of the peak positions of acid forms in the Soret region ( Table I)     The measurements were carried out at 20" as described in the text. Fluoride Titration-The Hill plots for the formation of fluoride complexes for various myoglobins (Fig. 5B) indicated that the affinity for fluoride is increased in the order of deutero-, meso-, proto-, monoformyl-monovinyl-, 2,4-diformylmyoglobin. The magnitude of affinity for fluoride was decreased by the factor of 300 compared with that of affinity for azide for all myoglobins studied in this experiment.
Cyanate and Thiocyanate Titrations-The Hill plots for cyanate and thiocyanate complexes formation are shown in Fig. 5, C and D. The slope for both complexes was 1 for all myoglobins examined. The affinities for cyanate and thiocyanate decreased in the order 2,4-diformyl-, monoformyl-monovinyl-, protomyoglobin.
However, meso-and deuteromyoglobins exhibited similar affinities for these ligands to that of protomyoglobin.

Effects of pH and Hemin Substitution on Dissociation Constants
(K)--The pK, and pK values of various myoglobins for several ionic ligand complexes are summarized in Table II. In all cases affinities (pK) for fluoride, thiocyanate, and azide at pH 6.0 were higher than at pH 7.0. As shown in Fig. 6, all myoglobins exhibited increasing ligand affinities with the increase of electron-withdrawing power of the porphyrin side chains, except for the affinities of deutero-and mesomyoglobins for fluoride, cyanate, and thiocyanate.

Properties
of Two Zsomeric Monoformyl-monouinylmyoglobins in Ferric Form-Significant differences between the absorption spectra of the two isomeric monoformyl-monovinyl ferric myoglobins were observed for all liganded states examined. The order and magnitide of the optical red shifts for the  three formylmyoglobins were similar to those for the ferrous states (3). The absorption maxima at the Soret region shifted to longer wavelengths in the increasing order of proto-, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, 2,4-diformylmyoglobin. The line shapes in the visible region were also different. Since the two isomeric free hemes have similar optical properties (a), these differences observed after recombination with apomyoglobin must be due to the interaction between heme and protein. In contrast, the ligand binding properties of these two isomeric ferric myoglobins were found to be similar. The pK values of the affinities for ligands and the pK, values of the acid-alkaline transition for the isomers were intermediate between those of proto-, and 2,4-diformylmyoglobins, suggesting that there is functionally little specificity in the two vinyl groups at positions 2 and 4 of the porphyrin ring, and that the total electron-withdrawing power of the substituents, regardless of the position, affects the affinity for ligand binding. Similarity in the pK, values of 2-vinyl-4-formyl-(8.02) and 2-formyl-4-vinyl-(7.95) myoglobins indicates that the ionization properties of the water molecules bound to the ferric irons, i.e. the electron densities of the iron atoms of these two isomeric myoglobins are similar.
Relation between pK, of Porphyrin and Ligand Affinity of Ferric Myoglobin-The close relationship between the electron-withdrawing power of the substituents at positions 2 and 4 of the porphyrin ring and the change in the reactivities of the ferric forms of horseradish peroxidase and sperm whale myoglobin were reported by Makino and Yamazaki (11,12). Similar relationships were observed for horse heart myoglobin between the pK, of the acid-alkaline transition or pK of the azide complex formation and pK, of the metal-free porphyrins (Fig. 6.). The dissociation constants (K) for the other ligands, The plots are acid-alkaline titration (01, complex formation of azide at pH 6.0 (A), thiocyanate at pH 6.0 (O), cyanate at pH 7.0 (A), and fluoride at pH 6.0 (0) of the ferric myoglobins having hemes with various side chains at positions 2 and 4 of the porphyrin ring. All data were obtained at 20" fluoride, cyanate, and thiocyanate, however, deviated from straight lines (Fig. 6). Although the reason for this deviation is not clear, it may be related to the thermal equilibrium state of the ferric heme iron. As mentioned below, the acid form of ferric formylmyoglobin exhibits a thermal mixture of the high spin state and low spin state spectra (24) at room temperature, whereas those of ferric proto-, deutero-, and mesomyoglobins show the typical high spin spectra (25)(26)(27).
Optical and Spin State Properties of Ferric Formylmyoglobins-The substitution of vinyl groups by formyl groups significantly changes the optical properties of ferric myo-globins. The acid forms of the three ferric formylmyoglobins give the four-banded spectra of a mixed type in the visible region at pH 6.0 (Fig. lA). Two peaks at about 510 and 660 nm of these four peaks are the so-called charge transfer bands and are specific to the high spin state. The other two peaks at about 560 and 600 nm appear close to the wavelengths where the alkaline form of ferric myoglobin has its maxima and are attributed to the spectra of the low spin state. Since there is no spectral change when pH is lowered from 6 to 5, the acid form spectra of the formylmyoglobins are undoubtedly those of a mixture of high and low spin forms, which may be induced by the strong electron-attractive formyl groups at the periphery of the porphyrin ring. Iizuka and Orii (24) reported that at a low temperature of 89 K, the acid form of chlorocruoromyhglobin (complex of sperm whale apomyoglobin and 2-formyl-4-vinyldeuterohemin) in Tris-HCl buffer, pH 7.0, exhibited the high spin type of spectra with less distinct bands at 550 and 600 nm, which are due to the low spin state form. However, the acid form of the other reconstituted myoglobins with less electronattractive side chains, such as meso-and deutero-derivatives, have typical high spin-type spectra similar to that of protomyoglobin (9). The dissociation constants of meso-, deutero-, and protomyoglobins for the complexes of fluoride, cyanate, and thiocyanate, which are of high spin types, are similar to one another, whereas the constants of the formylmyoglobins vary according to the pK, values of the porphyrins. Because such deviation is not found when the ligands are azide and hydroxyl ion, which form the complexes of the low spin state, there may exist a certain correlation between the spin state and the ligand affinity of ferric hemoprotein.