Heme-linked protonation of HCN, CO, NO and O2 complexes of reduced horseradish peroxidases

ARCHIVES

OF

BIOCHEMISTRY

Heme-Linked

AND

Division,

171,

737-744

(19%)

Protonation of HCN, CO, NO and 0, Complexes Reduced Horseradish Peroxidases HIROKAZU

Biophysics

BIOPHYSICS

Research

YAMADA’ Znstitute

of Applied Received

AND

ISA0

Electricity, May

of

YAMAZAKI Hokkaido

University,

Sapporo,

Japan

27, 1975

Small reversible changes in the absorption spectra of HCN, CO, NO and 0, complexes of ferrous diacetyldeuteroperoxidase A, not hitherto observed, were attributed to proton dissociation of a distal amino acid residue. From spectrophotometric titration data the pKa was measured as 5.5 (HCN), 5.6 (ligand free), 6.0 (CO), 6.55 (NO) and 8.0 (0,). The value of 8.0 for the pKa of the 0, complex was also obtained from a curve of pH dependence of proton uptake in the reaction of the ferrous enzyme with 0,. Absorption bands in the visible region were shifted to longer wavelengths in the order of CO to NO to 0, which is the decreasing order of the energy of m* level of these diatomic ligands. The pKa values for CO complexes of ferroperoxidases, isoenzymes A and (B+C) were varied with substituents at the 2 and 4 positions of deuterohemin IX, and the ApK$ApK, ratio was about 0.3 in both series of isoenzyme preparations, where pK, is a measure of basicity of pyrrole nitrogen. The present data support the previous conclusion (Yamada and Yamazaki (1974) Arch. Biochem. Biophys. 165, 728) that the fla for ferroperoxidases, measured from small reversible changes in the absorption spectra, represents a proton dissociation constant of a distal amino acid residue and that there is hydrogen bonding between the residue and a ligand atom directly bound to the iron atom.

Heme-linked groups have received considerable attention because of their possible participation in the function of hemoproteins (1). The presence of a heme-linked group with fla = ca. 7 in horseradish ferroperoxidase was suggested by Theorell (1) and Harbury (2). From data of proton balance in conversions between five oxidation-reduction states of horseradish peroxidase it was suggested that the hemelinked group is a distal amino acid residue that is hydrogen bonded to the ligand at the sixth coordination position in the ferric and oxyferrous forms of the enzyme (3). In this paper we shall report regular changes in fla of the heme-linked group that occur when ferrous diacetyldeuteroperoxidase A unites with CO, NO and 0, and also when various groups are substituted at the 2,4-positions of deuteroporphyrin IX in the ferroperoxidase-CO complex.

MATERIALS

131 0 1975 by Academic Press, Inc. of reproduction in any form reserved.

METHODS

Horseradish peroxidase was purified from wild horseradish roots by the method of Shannon et al. (4) with slight modification. The enzyme preparations used in this experiment were horseradish peroxidase (B+C), a mixture of isoenzymes B and C, and horseradish peroxidase A, a mixture of isoenzymes A, and AZ, according to nomenclature used by Paul (5) and Shannon et al. (4). Preparation of artificial peroxidases has been described previously (6, 7). The pH Stat and spectrophotometric titrations were carried out in a Dutton-type cuvette (8) with which both spectrophotometric and pH measurements could be done at the same time. The reaction temperature was 20°C except for experiments with oxyperoxidases. Anaerobic experiments were carried out under an N, atmosphere after a l-h draft of N, gas. Highly purified N, gas (99.9995%) obtained from a commercial source was used without further purification. Spectrophotometric measurements were performed with a Hitachi recording spectrophotometer, Model 124, equipped with a cuvette compartment thermostatically controlled, and pH Stat titrations were performed with an automatic pH Stat titration assembly of Radiometer (Copenhagen), combined an

1 Present address: Central Research Laboratory, Kanebo Co., Ltd., 1-chome Tomobuchicho, Miyakojima, Osaka. Copyright All rights

AND

738

YAMADA

SBR 2 C titrigraph 11 autoburette.

with

a TIT2

titrator

AND

and an ABU

YAMAZAKI HCN

CO

NO

RESULTS

Like hemoglobin and myoglobin, ferroperoxidases react with CO (1, g-19>, NO (20, 21)and 0, (20, 22, 23) to form their corresponding derivatives. Despite many experimental data so far reported for these derivatives, pH dependence of their optical absorption spectra has never been described. Figure 1 shows that there are small but distinct changes in their visible spectra between acid and alkaline forms of the HCN, CO or NO complex of ferrous diacetyldeuteroperoxidase A. Though these changes in absorbance are very small, typical proton dissociation curves were obtained by careful spectrophotometric titrations with acid and alkali (Fig. 2). Slight discrepancies between forward and backward titration curves in the cases of the CO and NO complexes appeared to be due

450

500

550 Wavelength

600

650

( nm 1

FIG. 1. The effect of pH on the visible spectra of HCN, CO and NO complexes of ferrous diacetyldeuteroperoxidase A. From the top: HCN complex at pH 7.44 (solid line) and pH 5.00 (dotted line); CO complex at pH 8.95 (solid line) and pH 4.95 (dotted line); and NO complex at pH 8.55 (solid line) and 5.20 (dotted line). The HCN complex was formed in the presence of 100 mM KCN, 52 pM enzyme and a slight excess of sodium dithionite. The CO complex was formed in the presence of 160 PM CO, 34 PM enzyme and a slight excess of sodium dithionite. The NO complex was formed in the presence of 29 PM enzyme, 1 mM NaNO, and 2 mM sodium dithionite. The experiments were performed under anaerobic conditions in the presence of 0.1 M KCl. The absorbance scales for the CO and HCN complexes are less than scale shown on the ordinate by 0.1 and 0.2, respectively.

5

6

7

8

PH

FIG. 2. Spectrophotometric titrations of HCN, CO and NO complexes of ferrous diacetyldeuteroperoxidase A with acid and alkali. The forward (circles) and the backward (triangles) titrations were carried out with 0.5 N HCl and 0.5 N NaOH, respectively. Other experimental conditions were the same as described in the legend of Fig. 1. The midpoint pH’s were 5.5, 6.0 and 6.5 for the HCN, CO and NO complexes, respectively.

to experimental errors but not to instability of the complexes. At any rate, the proton dissociation constants could be measured from these curves and are listed in Table I. Ferrous diacetyldeuteroperoxidase A reacted with 0, to form a compound which gave an absorption spectrum typical of oxyperoxidase (Fig. 3). It has been reported that the oxyperoxidase decomposes into the ferric enzyme without any appreciable intermediate (20, 22, 23). The oxy-form of diacetyldeuteroperoxidase A was much more stable than that of the native enzyme and the half-decay time of oxydiacetyldeuteroperoxidase A was about 5 h at 20°C. Even in this case it was difficult to prepare a solution containing 100% oxyperoxidase. Under aerobic conditions the oxyperoxidase solution contained a small amount of ferriperoxidase. As the absorption spectrum of ferriperoxidase markedly varied with pH, the spectrophotometric titration of the oxyperoxidase with acid and base was performed in the presence of cyanide. For the ferric diacetyldeuteroperoxidase A, the pKa for ionization to its alkaline form and the dissociation constant for its cyanide complex are reported to be 7.7 (6) and 0.1 PM (7), respectively. Consequently, in the presence of 0.1 mrvr cyanide almost all of the ferric enzyme existed as the cyanide complex even at pH 10. Absorbance of the cyanide complex at 586 nm varied slightly with pH, but it could be

HEME-LINKED TABLE

I

SPECTRAL DATA AND PROTON DISSOCIATION CONSTANTS FOR COMPLEXES OF FERROUS DIACETYLDEUTEROPEROXIDASE A Ligand

A maxa(nm)

PKa

HCN

a-Band

p-Band

576 580 582 586

546 -

5.5 5.6 6.0 6.55 8.0

co NO Gb

547 550 551

a The values of A,,, for the CO and NO complexes were obtained by resolving the visible spectra into two bands. The spectra of acid forms were used for calculation. * Experiments at 7°C.

739

PROTONATION

ured to be 8.0. A similar titration experiment revealed the presence of a hemelinked group with pKa = 8.60 in the cyanide complex of the ferric enzyme (Fig. 4). It was reported that uptake of one proton occurred when one ferroperoxidase molecule in the deprotonated state reacted with 0, to form the oxyenzyme (3). The pKa for the heme-linked group of ferrous diacetyldeuteroperoxidase A was found to be 5.6 (24). In order to confirm the relation between two heme-linked groups with pKa = 5.6 in the ferrous enzyme and with pKa = 8.0 in the oxyenzyme, the amount of proton uptake caused by the reaction between the ferroperoxidase and 0, was measured at various pH values. Some complication was added to the analysis of the results by the fact that in the reaction with 0, about 90% of the ferroperoxidase was converted into the oxyenzyme but the remainder was oxidized to the ferric enzyme which has an alkaline ionization group of pKa = 7.7. In Table II, corrections were made according to the following equations. Ferroperoxidase

+

ferriperoxidase 0’.

0 500

’ 550 Wavelength

’ 600

650

Cm-n)

FIG. 3. The effect of pH on the visible spectra of ferrous-O, and ferric-cyanide complexes of diacetyldeuteroperoxidase A. The ferrous-O, complex was prepared by the addition of 0.5 ml of an O,-saturated solution to a 5 ml solution containing 54 qf ferrous enzyme, 1 pM ferric enzyme and 0.1 M KCl. The spectra were measured at pH 10.00 (solid line) and pH 5.75 (dotted line) in the presence of 100 PM KCN. The experiment was carried out at 7°C. The spectra of the cyanide complex were measured at pH 9.45 (solid line) and pH 5.97 (dotted line) with a solution containing 36.5 PM ferric enzyme, 10m4 M KCN and 0.1 M KC1 at 20°C.

concluded that the spectrophotometric titration curve, obtained with the oxyperoxidase solution containing the cyanide complex present at less than 10% of the total enzyme, was attributed to a heme-linked protonation of the oxyperoxidase (Fig. 4). The titration experiment was carried out at 7°C to avoid decomposition of the oxyperoxidase. The pKa value was thus meas-

+ H+(K,.) +

l/40,

+ ‘/zH,O

+ H+(K,,),

where H+(K,)I(H+), = KACK,. + H+); (H+(K,)I (H+jO = K&K,, + H+); K, and K,, stand for proton dissociation constants of hemelinked groups of the ferrous and ferric enzymes, respectively; and (H+), is the con-

407 6

7

8 PH

FIG. 4. Spectrophotometric titrations of ferrous0, and ferric-cyanide complexes of diacetyldeuteroperoxidase A with acid and alkali. The forward titrations (circles) were carried out with 0.5 N HCl (ferrous-0,) and 0.5 N NaOH (ferric-cyanide), and the backward titrations (triangles) with 0.5 N NaOH (ferrous-o,) and 0.5 N HCl (ferric-cyanide). Other experimental conditions were the same as described in the legend of Fig. 3. The smooth curves are theoretical with fla values of 8.0 for the ferrous-O, complex and 8.6 for the ferric-cyanide complex.

740

YAMADA

AND YAMAZAKI TABLE

UPTAKE PH

Total enzyme (PM)

OF PROTONS

IN THE

FelTOIl. enzyme” (PM)

REACTION Addition

oxyform (PM (B)

6.00 7.00 7.40 7.70 8.00 8.25 8.50 8.75 9.00

39.0 42.0 38.5 33.5 40.0 36.5 40.0 38.5 56.0

31.2 33.6 29.5 25.0 32.5 27.8 30.3 32.0 48.0

29.0 32.0 27.0 22.5 29.0 26.5 28.0 29.0 42.0

2nd additionb H+ uptake (5mM HCl) (/a

H+ uptake 6mM HCI) (rl)

29.5 47.2 25.5 14.1 26.6 12.2 10.7 6.9 -2.7

(1The enzyme was reduced by addition of dithionite, free of both 0, and dithionite. b Blank tests. c Calculated as described in the text.

centration of protons normalized to that of ferriperoxidase formed during the reaction with 0, (corresponds to A - B in Table II). It was then possible to calculate the amount of protons released in the oxidation of ferroperoxidase to the ferric enzyme by 0, at each pH. Finally, the amount of proton uptake during the formation of the oxyenzyme from ferroperoxidase and 0, was calculated as shown in the last two columns of Table II. These values are plotted against pH in Fig. 5. This result shows that one proton was incorporated into the enzyme upon binding of 0, at pH 7 but no proton uptake occurred in an alkaline pH range. Although the experimental data were scattered over a range, the pKa of the heme-linked group was measured as about 8.0. This value was consistent with that obtained from the spectrophotometric titration data illustrated in Fig. 4. It was found that the effect of electronwithdrawing capacities of 2,4-substituents upon proton dissociation constants of heme-linked groups are quite different between ferri- and ferroperoxidases (24). The ratios of ApKJApK, and ApKdApK, are reported to be 0.1 and 1.0, respectively.

A

DIACETYLDEUTEROPEROXIDASE

of 0.3 ml of 0, solution

1st addition

(A)

II

OF FERROUS

7.6 15.9 0.2 0 12.2 3.9 -1.5 1.0 -3.1

Proton H+ uptake A (/AM) m

uptake reactions

Ferrous -+ ferric’ (PM) CD)

21.9 31.3 25.3 14.1 14.4 8.3 12.2 5.9 0.4

1.5 1.3 1.7 1.3 1.2 0.3 0.3 0.2 0.2

due of

WITH

0,

to

C-D B

Ferrous + oxy-form (PM) (‘2 - D)

20.4 30.0 23.6 12.8 13.2 8.0 11.9 5.7 0.2

and at this stage the solution (5 ml, 0.1

0.70 0.94 0.88 0.57 0.46 0.30 0.43 0.20 0.005 M

KCU was

Here, K, is the proton dissociation constant for monocationic species of the metal-free porphyrin and is a measure of the basicity of the pyrrole nitrogen. It appeared to be of particular importance to investigate the relationship between pK, and pK,.(CO), where K&CO) stands for proton dissociation constant of a heme-linked group in the ferroperoxidase-CO complexes. Values of the pK,(CO) obtained from the spectrophotometric titration experiments (similar to Fig. 2) are plotted against pK, in Fig. 6. These plots are somewhat dispersed from straight lines but it

01.

6

7

8

9

PH

FIG. 5. The effect of pH on proton balance in the of ferrous diacetyldeu.teroperoxidase A with 0, to form the oxyperoxidase. The smooth curves are theoretical with pKa values of 5.6 and 8.0 (see the text). Plotted from data in Table II. reaction

HEME-LINKED

DlacefylmAem

5

' 3

Chlorocr”oro

Pro,0

Deuft-fO

A,

ba 4

Meso

i,L 5

6

P'G

FIG. 6. Dependence of pK, and pK,(CO) upon pK,. Data for pK, (triangles) are from Yamada et al. (24). For fl,(CO), the data in Table III are plotted (circles). The values of pK, for diacetyldeutero-, chlorocruoro-, proto-, deutero-, and mesoporphyrins are indicated by arrows.

was concluded that the ratio of ApKJCO) to ApK, was about 0.3 and values of pK,(CO) for ferrous deuteroperoxidaseCO complexes were exceptionally low in both isoenzyme series. The anomaly in the relationship between pK, and reactions of the hemoprotein has frequently appeared in the case of deuterohemoproteins (7, 19). DISCUSSION

The distal amino acid residue, though not directly bonded to the heme, has been considered to be significant for modes of reaction of various hemoproteins (3, 16, 24-34). It seems reasonable that Theorell (1) used the term “heme-linked group” in a wide sense. Spectrophotometric methods have been used most frequently for the determination of the nature of hemelinked groups. However, except for alkaline ionization of ferric hemoproteins which is accompanied by a large change in absorption spectra, only a few data have been reported regarding other pH-dependent changes in the absorption spectra of these proteins (3, 24, 31, 35-37). In these cases the spectral changes are very small and it seems rather difficult to determine the nature of the proton dissociation data groups from spectrophotometric alone. The heme-linked groups with pK, = 5.8 for peroxidase A and 7.3 for peroxidase (B+C) have been assigned as the distal amino acid residue from measurements of

PROTONATION

741

proton uptake or release in reactions of the peroxidases (3) and of their redox potentials (24) under various experimental conditions. The effect of 2,4-substituents upon the pK,(CO) value also suggests the presence of a weak interaction between the distal base and the ligand. The ratio of ApK,(CO) to ApK, may be regarded as a measure of the strength of the interaction. The results demonstrated in Fig. 6 are interpreted as follows. The proton on the distal base dissociates without an influence of electron density on the iron atom in the case of ligandfree ferroperoxidase. When CO is coordinated at the sixth position the effect of electron density on the iron atom upon the distal base is accentuated through 7r-donation from the iron to the ligand. Accordingly, the ratio of ApK, for the 0, complex to ApK, is expected to be much higher than 0.3. The experimental confirmation, however, does not appear to be feasible. As the ApK,,lApK3 ratio is about 1.0 for both peroxidase preparations, isoenzymes A and (B+C) (71, it can be concluded that electronic interaction between the distal base and the iron atom is strong in the ferriperoxidases. Studying electron paramagnetic resonance (epr) and electronic spectra, Wayland et al. (38) have proposed a molecular orbital model for complexes of cobalt (11) porphyrin with CO, NO, and 0, in two cases of linear and bent adducts of these diatomic molecules. It seems likely, however, that for complexes of a hemoprotein with these ligands their bondings are expected to be identical in orientation because of interactions with the protein. As suggested by Wayland et al. (38) one important difference between bondings of these ligands will come from the fact that the energy of the 7~*level decreases monotonically from CO to NO to 0,. According to the theory, the pKa shift that occurs upon complexing with these ligands can be interpreted in terms of a difference in electron transfer from the iron atom to the ligand. It seems likely that the change in basicity of the distal amino acid residue is caused not only by a direct electrostatic interaction but also by a hydrogen bonding be-

742

YAMADA

AND

tween the base and the ligand. It should be noticed here that the spin state of the heme iron may not have an influence on the pK, shift. A decrease in the pKa upon complexing with cyanide will be expected if one assumes that only the proton-dissociated form of the cyanide complex is stabilized through a hydrogen bond, as shown schematically in Fig. 7. From the above consideration it is also suggested that the effective oxidation state of the iron atom is increased from two to three in the order of CO, NO and 0,. For ferrohemoprotein-0, complexes the formalism Fe(III)-O,has been proposed by several workers (3, 32, 39-43). This model may not be incompatible with the data of infrared spectroscopy (44, 45). In general, however; it is difficult to determine the exact effective oxidation state of the iron atom. Smith and Williams (46) have presented

FIG. 7. Model of proton the heme site. The values diacetyldeuteroperoxidase

SPECTRAL

DATA

dissociation occurring at of pIfa indicated are for A.

AND PROTON

TABLE DISSOCIATION CONSTANTS DEUTEROPEROXIDASES

Complexes

A group Meso 2,4-CH,CH, Deutero: 2,4-H Proto (natural): 2,4-CH=CH, Chlorocruoro: 2-CHO, 4-CH= Diacetyldeutero: 2,4-CO-CH, (B+C) group Meso Deutero Proto (natural) Chlorocruoro Diacetyldeutero a The obtained

YAMAZAKI

an exhaustive review of the extensive literature on hemoprotein spectra. The effect of ligands on the spectra are discussed mostly in cases of ferric hemoproteins. Spectral data obtained in this experiment are concerned with ferroperoxidases as shown in Tables I and III. Though the data are not yet sufficient, there is good correlation between the position of the CY-or p-peak and the shift of pKa of the distal base except for the a-peak of chlorocruoroperoxidase-CO complexes. The effect of 2,4-substituents upon the absorption peaks is similar to those reported previously (6, 7, 18, 47-49). Schonbaum et al. (50) have suggested the existence of an acid-base interaction between a ligand and a distal amino acid residue in reactions of acetylated horseradish peroxidase. The present results will provide other evidence to support the idea. The results also indicate that the nature of bonding of CO and 0, to ferroperoxidases differs from the case of myoglobin. No significant change in proton dissociation has been observed in reactions of myoglobin with 0, and CO (36, 51). We consider that this fact is related to our presumption that the hydrogen bond between the distal histidine and a ligand is not of significance in complexes of myoglobin with 0, and CO (24). This idea, however, contrasts with the conclusion drawn by Yonetani et al. (32) from data on cobalt III FOR FERROUS-CO A AND (B+C) K,(CO)

CH,

COMPLEXES

OF 2,4-SUBSTITUTED

o-Band

A maXe bm) p-Band

6.90 6.40 6.72 6.15 6.0

562 560 570 596 580

533 528 540 (5751 548 547

8.2 7.9 8.1, 8.25 7.6 7.35

563 558 571 598 581

533 528 540 (5751 545 547

values of A,,, for complexes of deutero-, chlorocruoro-, and diacetyldeuteroperoxidases by resolving the spectra into components. The spectra of the acid forms were used throughout.

were

HEME-LINKED

PROTONATION

hemoproteins. The problem should be solved by further experiments. Comparative studies along this line will be reported elsewhere 62). ACKNOWLEDGMENT Unnatural Ryu Makino

hemes to whom

H.

(1947)

Aduan.

by

Dr.

26. Enzymol.

7, 265-

303. 2. HARBURY, H. A. (1957)J.Biol. Chem. 225,10091024. 3. YAMADA, H., AND YAMAZAKI, I. (1974) Arch. B&hem. Biophys. 165, 728-738. 4. SHANNON, L. M., KAY, E., ANDLEW, J. Y. (1966) J. Biol. Chem. 241, 2166-2172. 5. PAUL, K. G. (1958) Acta Chem. &and. 12,13121318. 6. MAKINO, R., AND YAMAZAKI, I. (1972) J. Biothem. (Tokyo) 72, 655-664. 7. MAKINO, R., AND YAMAZAKI, I. (1973) Arch. Biothem. Biophys. 157, 356-368. 8. DUTTON, P. L. (1971) Biochim. Biophys. A& 226, 63-80. 9. THEORELL, H. (1943) Ark. Kemi Mineral. Geol. A 16, No. 14. 10. KEILIN, D., AND HARTREE, E. F. (1951) Biochem. J. 49, 88-104. 11. CHANCE, B. (1952) J. Biol. Chem. 197,577-589. 12. KEILIN, D., AND HARTREE, E. F. (1955) Biochem. J. 61, 153-171. 13. MORITA, Y. (1956) Mem. Res. Inst. Food Sci., Kyoto Univ. 11, 38-48. 14. KERTESZ, D., ANTONINI, E., BRUNORI, M., WYMAN, J., AND ZITO, R. (1965) Biochemistry 4, 2672-2676. 15. WI~ENBERG, B. A., ANTONINI, E., BRUNORI, M., NOBLE, R. W., WI’ITENBERG, J. B., AND WYMAN, J. (1967) Biochemistry 6, 1970-1974. 16. RICARD, J., AND NARI, J. (1966) Biochim. Biophys. Acta 113, 57-70. 17. BLUMBERG, W. E., PEISACH, J., WITTENBERG, B. A., AND WITTENBERG, J. B. (1968) J. Biol. Chem. 243, 1854-1862. 18. TAMURA, M., ASAKIJRA, T., AND YONETANI, T. (1972)Biochim. Biophys. Actu 268,292-304. 19. MAKINO, R., AND YAMAZAKI, I. (1974)Arch. Biothem. Biophys. 165, 485-493. 20. WITTENBERG, J. B., NOBLE, R. W., WITTENBERG, B. A., ANTONINI, E., BRUNORI, M., AND WYMAN, J. (1967) J. Biol. Chem. 242,626-634. 21.

22.

YONETANI, LEIGH,

T.,

YAMAMOTO,

24.

25.

were kindly supplied we are much indebted.

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