Azide: The Effect of Heme-Linked Ionizations on the Rate of Complex Formation

Archives of Biochemistry and Biophysics Vol. 362, No. 1, February 1, pp. 148 –158, 1999 Article ID abbi.1998.0991, available online at http://www.idealibrary.com on

Metmyoglobin/Azide: The Effect of Heme-Linked Ionizations on the Rate of Complex Formation 1 Josephine Lin, James Merryweather, Lidia B. Vitello, and James E. Erman 2 Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115

Received August 14, 1998, and in revised form October 19, 1998

The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN 3, binds to metmyoglobin with a rate constant of (3.8 6 1.0) 3 10 5 M 21 s 21. Protonation of a group with an apparent pK a of 4.0 6 0.3 increases the rate of HN 3 binding 6.5-fold to (2.5 6 0.8) 3 10 6 M 21 s 21. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N 32, binds to metmyoglobin with a rate constant of (4.7 6 0.3) 3 10 3 M 21 s 21, about two orders of magnitude slower than HN 3. Conversion of aquometmyoglobin to hydroxymetmyoglobin slows azide binding significantly. Binding of HN 3 to hydroxymetmyoglobin cannot be detected, while N 32 binds to hydroxymetmyoglobin with a rate of 5.7 6 3.2 M 21 s 21, almost three orders of magnitude slower than N 32 binding to aquometmyoglobin. Protonation of the distal histidine facilitates HN 3 dissociation from the complex. HN 3 dissociates from the metmyoglobin/ azide complex with a rate constant of 18 6 6 s 21, while the azide anion dissociates with a rate constant of 0.16 6 0.02 s 21, about 100 times slower. The apparent pK a for His-64 is essentially the same in metmyoglobin and the metmyoglobin/azide complex, 4.0 6 0.3 and 4.4 6 0.2, respectively. The ionic strength dependence of the observed association rate constant is influenced by both primary and secondary kinetic salt effects. The primary kinetic salt effect is anomalous, with the rate of N 32 binding decreasing with increasing ionic strength above the isoelectric point of metmyoglobin where the protein has a net negative charge. The ionic strength dependence of the dissociation rate constant can be described solely in terms of the ionic strength 1 This work was supported in part by grants from the NSF (MCB 95-13047) and from the NIH (R15 GM54328-01). 2 To whom correspondence should be addressed. Fax: (815) 7534804.

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dependence of the acid dissociation constant for His-64 in the metmyoglobin/azide complex, a secondary kinetic salt effect. © 1999 Academic Press

Ferric heme proteins bind small, weakly basic ligands such as fluoride, azide, cyanide, and imidazole. There are substantial differences in the binding rates and binding affinities for these types of ligands to various heme proteins. These differences reflect the diverse chemical environments found in the active sites of heme proteins and can provide insight into the structural basis for differential reactivity of the many heme proteins found in living organisms. Essentially all of the ligands which bind to the Fe(III) state of heme proteins can exist in two or more forms, differing in charge and the number of protons bound. One of the classic problems in heme protein chemistry is to determine the relative reactivities of neutral and charged ligands in binding to the Fe(III) heme. Determination of the pH dependence of the association and dissociation rate constants for ligand binding gives information on the relative reactivities of the various protonated forms of the ligand. However, interpretation of the data is not straight forward since most heme proteins have ionizable groups in the vicinity of the heme which can modulate protein reactivity. It is often difficult to sort out the effect of heme-linked ionizations in the protein from the effect of ligand ionization. One of the most thoroughly investigated ligand binding reactions is the binding of azide to metmyoglobin (1–11). Despite these studies, there is disagreement as to the mechanism of azide binding. Czerlinski and coworkers (3– 6) conclude that hydrazoic acid, HN 3, is 300 times more reactive than the azide anion, N 32, and that various protonated forms of metmyoglobin have relatively little effect on the rate of binding (5, 6). Alberty and co-workers (7, 8) come to the opposite 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

METMYOGLOBIN/AZIDE KINETICS

conclusion, that the N 32 is the most reactive form of the ligand and that heme-linked ionizations have major impact of the reactivity of the protein. Alberty attributes the pH dependence of the association rate constant to two heme-linked ionizations in the protein, one with a pK a of 4.7 and one with a pK a of 5.7. The Alberty group suggest that the group with a pK a of 4.7 is a heme propionate and that the group with the pK a of 5.7 is the distal histidine. In light of current information on the pK a values of the ionizable groups in myoglobin (12), these assignments are incorrect. Friend et al. investigated equilibrium binding of azide to sperm whale metmyoglobin and calculated the electrostatic free energy contribution to the binding of azide for all charged groups in the protein (10). Friend et al. found that essentially all of the charged groups had some influence on the observed equilibrium constant. In this model every acidic or basic group in the protein can contribute to binding affinity of the azide anion and the pH dependence of the equilibrium constant is modulated by the state of ionization of each group and its distance from the heme site. In addition to studies of azide binding to wild-type sperm whale and horse metmyoglobin, a number of studies using myoglobin mutants have appeared in recent years (13–19). The most complete study is that of Brancaccio et al. (18), who investigated the effect of mutations in the distal heme pocket in sperm whale, pig, and human myoglobin on azide and cyanide binding. Brancaccio et al. constructed mutations at positions 29, 45, 46, 64, 67, and 68 within the distal pocket. Almost all mutations increased the rate of azide binding with the largest effects, ;10 3, occurring upon converting either His-64 or Phe-46 to amino acid residues with small apolar side chains. Both types of mutations are consistent with the proposal that His-64 controls access to the distal heme pocket through a gating mechanism (18, 20). We have recently investigated the binding of fluoride to horse metmyoglobin over an extended pH range (21). Two heme-linked ionizations in the protein affect the association rate. One, with an observed pK a of 4.4 6 0.5, is attributed to the distal histidine and the second is due to formation of hydroxymetmyoglobin. The association rate for fluoride binding is dominated by diffusion of HF into the distal heme pocket followed by proton dissociation and binding of the fluoride anion to the heme iron. Protonation of the distal histidine increases the rate of HF binding by about a factor of three, suggesting two compensating roles. Protonated His-64 provides open access to the distal heme pocket (20) while unprotonated His-64 acts as a base, promoting proton dissociation from HF and binding of the fluoride anion to the heme iron (21). Due to the uncertainty in the mechanism of azide binding, whether HN 3 or N 32 dominates the reaction

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(3– 8), the various assignments of the heme-linked ionizations (8, 12), and the multiple roles of His-64 in ligand binding (21), we decided to investigate azide binding to metmyoglobin over as large a range of pH as feasible. The data at the pH extremes are the most informative in terms of the reactivities of hydrazoic acid, the azide anion, and various protonated states of metmyoglobin. We have used pH-jump techniques to measure the kinetics of metmyoglobin–azide complex formation between pH 3.5 and 11.5. This pH range encompasses the ionization of hydrazoic acid (pK a 5 4.5), His-64 (pK a 5 4.0 6 0.3, this study), and the heme-bound water in metmyoglobin (pK a 5 8.9). We have also investigated the reaction as a function of ionic strength to help discriminate between reactions involving neutral HN 3 and negatively charged N 32. We conclude that HN 3 binds to metmyoglobin about 80 times faster than N 32 and that both the ionization of the distal histidine and ionization of the heme-bound water have significant effects on the observed rate of binding. Binding of HN 3 dominates the rate of complex formation below pH 6.5, while binding of N 32 to aquometmyoglobin is the dominant reaction at pH $ 6.5. MATERIALS AND METHODS Horse metmyoglobin and purified sodium azide were purchased from Sigma Chemical Co. (St. Louis, MO) and Fisher Scientific (Chicago, IL), respectively. Reagent grade chemicals were used for buffer preparation as follows: potassium acid phthalate, pH 3.5 to 4.0; potassium acetate, pH 4.0 to 5.5; potassium phosphate, pH 5.5 to 8.0 and pH 10.75 to 11.5; tris(hydroxymethyl)aminomethane, pH 8.0 to 9.5; and glycine, pH 9.5 to 11. The buffering ions were generally maintained at 0.01 M and the ionic strength adjusted by the addition of KNO 3. The contribution of the azide ion to the ionic strength was taken into account for all solutions. Concentrated stock solutions of metmyoglobin were prepared by dissolving lyophilized protein in a KNO 3 solution of appropriate ionic strength. The pH of this solution was ;pH 7. Small aliquots of the stock solution were dissolved into appropriate buffers for the spectroscopic and kinetic measurements. The concentration of metmyoglobin solutions was determined spectrophotometrically using an extinction coefficient of 188 mM 21 cm 21 at the Soret maximum for aquometmyoglobin (22). Spectra were determined using a Hewlett– Packard Model 8452A diode array spectrometer, a Cary Model 3E, or Cary Model 219 UV–visible spectrophotometer. Kinetic experiments were carried out by stopped-flow techniques. Over the course of the study three different instruments were used: a Durrum-Gibson Model D-110, a Hi-Tech Model SPQ-51, and an Applied Photophysics, Ltd., Model SX.17MV. Metmyoglobin is stable between pH 5 and 11.5 and in this pH region buffered metmyoglobin was placed in one drive syringe of the stopped-flow spectrometer and rapidly mixed with an equal volume of buffered azide solution. Reactions were carried out under pseudo-first-order conditions with the azide concentrations at least 10 times greater than that of metmyoglobin. For pH-jump experiments below pH 5.0, where metmyoglobin is unstable, metmyoglobin was dissolved in 0.10 M KNO 3 (pH ;7) and placed in one of the drive syringes. This was mixed in the stopped-flow with equal volumes of either potassium acetate or potassium hydrogen phthalate buffers, pH 3.5 to 4.75, containing up to 10 mM azide and enough KNO 3 to bring the final reaction mixture to

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When the ligand concentration is much higher than the protein, the concentration of free ligand remains essentially constant throughout the reaction and the observed rate constant is given by k obs 5 k a@L# 1 k d

FIG. 1. Spectrum of horse metmyoglobin (thin line) and the metmyoglobin/azide complex (thick line). The visible region of the spectrum is also shown expanded by a factor of five. The principal bands of the metmyoglobin/azide complex (a, b, and Soret) occur at 579, 541, and 420 nm, respectively. constant ionic strength of 0.10 M. The pH of the solution was measured after the reaction took place. All reactions were carried out at 25°C. The apparent pK a for hydrazoic acid at 25°C was determined potentiometrically between 0.03 and 1.0 M ionic strength according to the method of Boughton and Keller (23). Weighed samples of sodium azide (typically ;0.01 M), dissolved in various concentrations of KNO 3, were titrated with standardized HCl (;1.0 M). Plots of the volume of titrant versus pH were corrected for blank titrations and apparent pK a values calculated from the tabulated data. Calculated pK a values were reproducible to 60.02 units.

[2]

Both the association rate constant, k a, and the dissociation rate constant, k d, can be determined from a plot of k obs versus the ligand concentration (Fig. 2). At each condition of pH and ionic strength, k obs was determined at a minimum of five different ligand concentrations. At each ligand concentration, k obs was the average of at least three determinations. Values of k a and k d, along with estimates of their standard deviation, were determined by weighted linear least-squares regression according to Eq. [2]. The relative percentage of errors for k a and k d are 16 and 12%, respectively. Replicate experiments were performed on different days for about one-fourth of the experimental conditions. The standard deviations for k a and k d from replicate experiments averages 20% of the mean for k a and 22% for k d, giving a more realistic estimate of the day-to-day reproducibility of the data. pH dependence of k a and k d for the azide/metmyoglobin complex. We have measured the rate of azide binding to metmyoglobin between pH 3.5 and 11.5. Figure 3 shows a plot of the logarithm of the association rate constant, k a, as a function of pH. The value of k a decreases by about five orders of magnitude between pH 3.5 and 11.5. Two inflections in the plot of the logarithm of k a versus pH indicate that heme-linked ionizations in the protein influence the rate of binding.

RESULTS

Spectrum of the metmyoglobin/azide complex. Figure 1 shows the spectrum of horse metmyoglobin and the metmyoglobin/azide complex at pH 6.0. Binding of azide converts hexacoordinate, high-spin aquometmyoglobin into a mixed-spin azide complex. The low-spin component of the azide complex causes the red shift of the Soret band, an increase in absorptivity in the a and b bands at 580 and 540, respectively, and a decreased absorptivity in the charge transfer bands at 630 and 500 nm (Fig. 1). Kinetics of metmyoglobin/azide complex formation. Reversible complex formation between azide and metmyoglobin is illustrated in Eq. [1], where L represents the total ligand, i.e., the sum of HN 3 and N 32, and P represents the total protein, metmyoglobin. ka

P1L | 0 PL kd

[1]

FIG. 2. Observed pseudo-first-order rate constants for the reaction between metmyoglobin and azide as a function of the azide concentration. Weighted linear least-squares regression analysis to Eq. [2] of the text gives k a and k d values of (2.4 6 0.2) 3 10 4 M 21 s 21 and 1.1 6 0.1 s 21, respectively. Experimental conditions: pH 6.0, 0.01 M ionic strength, 25°C.

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METMYOGLOBIN/AZIDE KINETICS

Ionic strength dependence of the pK a for hydrazoic acid. In order to interpret the pH and ionic strength dependence of the azide/metmyoglobin reaction, the ionic strength dependence for the ionization of hydrazoic acid is required. Boughton and Keller, 1966 report pK°a values for hydrazoic acid at 20, 26, and 33°C and apparent pK a values between 0.00986 and 0.0427 M ionic strength at 20°C (23). We have interpolated their data to obtain apparent pK a values as a function of ionic strength at 25°C (Fig. 6). We have extended the work of Boughton and Keller by determining apparent pK a values between 0.03 and 1.0 M ionic strength (Fig. 6). There is good agreement between our data and that of Boughton and Keller. The data in Fig. 6 were fit to the equation

pK L 5 pK 0L 1 S

ÎI

~1 1 ÎI!

[4]

FIG. 3. pH dependence of the apparent bimolecular association rate constant, k a (solid circles), and the apparent dissociation rate constant, k d (solid triangles), for the binding of azide to horse metmyoglobin at 0.1 M ionic strength, 25°C.

The pH dependence of the dissociation rate constant is shown in Fig. 3. Between pH 4 and 5, binding of azide to metmyoglobin is much faster than metmyoglobin denaturation and both k a and k d can be obtained by plotting k obs versus ligand concentration. However, below pH 4 the rate of metmyoglobin denaturation and the apparent values of k d were about the same. It is not possible to deconvolute denaturation and ligand dissociation and values of k d are only reported between pH 4 and 11.5. The value of k d decreases by about two orders of magnitude over this pH range. Ionic strength dependence of k a and k d for the azide/ metmyoglobin complex. To assess the influence of electrostatic interactions on azide binding to metmyoglobin, the ionic strength dependence of the rate constants was determined at integral pH values between pH 5 and 10. Figures 4 and 5 show plots of the logarithm of k a and k d as a function of ionic strength. The data were fit to Eq. [3], where log k° is the logarithm of the rate constant at zero ionic strength, I is the ionic strength, and S is the slope of the plot.

log k 5 log k 0 1 S

ÎI ~1 1 ÎI!

[3]

Table I contains values for log k° and S for both k a and k d at integral pH values between pH 5 and pH 10.

FIG. 4. Ionic strength dependence of the apparent association rate constant, k a, at integral pH values between 5 and 10. The solid lines are the calculated ionic strength dependence of the rate constant using the mechanism shown in Scheme 1 and Eq. [5] of the text. Both secondary kinetic salt effects due to pK P1, pK P3, and pK PL and primary salt effects for k 023 and k 04 are required to fit the data.

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FIG. 6. Ionic strength dependence of the acid dissociation constant for hydrazoic acid at 25°C. The solid circles represent data from this study and the open circles are interpolated from the data of Boughton and Keller (23). The pK a for hydrazoic acid is denoted pK L to be consistent with nomenclature used in the mechanism of azide binding to metmyoglobin (Scheme 1).

DISCUSSION

FIG. 5. Ionic strength dependence of the apparent dissociation rate constant, k d, at integral pH values between 5 and 10. The solid lines are the calculated ionic strength dependence of the rate constant for the mechanism shown in Scheme 1 using Eq. [10]. The only parameter to depend upon the ionic strength in Eq. [10] is K C1.

where pK°L is the pK a value for hydrazoic acid at zero ionic strength and S is the slope of the plot. Best fit values for pK°L and S are 4.65 6 0.01 and 20.51 6 0.03.

TABLE I

Ionic Strength Dependence of k a and k d for the Metmyoglobin/Azide Reaction a Log k a 0 a

pH

Log k

5.0 6.0 7.0 8.0 9.0 10.0

5.15 6 0.03 4.49 6 0.02 4.11 6 0.02 3.80 6 0.03 3.19 6 0.03 2.45 6 0.04

a

Log k d 0 d

S

Log k

0.16 6 0.10 20.79 6 0.07 21.22 6 0.08 20.91 6 0.10 20.42 6 0.10 0.14 6 0.14

0.18 6 0.03 20.32 6 0.04 20.61 6 0.02 20.69 6 0.03 20.75 6 0.05 20.74 6 0.05

The data were fit to Eq. [3] of the text.

S 1.63 6 0.10 0.89 6 0.12 0.40 6 0.08 0.24 6 0.09 0.01 6 0.14 0.04 6 0.18

Mechanism of azide binding to metmyoglobin. Alberty and colleagues investigated the binding of a number of ligands to sperm whale metmyoglobin over a restricted pH range, generally between pH 5 and 8 (7, 8, 24 –26). The Alberty group assumed that binding of the anionic form of the ligand was the preferred reaction pathway and found that two ionizable groups in metmyoglobin influenced the rate of ligand association. These two groups have apparent pK a values of ;4.7 and ;5.7 in sperm whale metmyoglobin. Goldsack et al. suggest that the group with a pK a of 4.7 is a heme propionate and the group with pK a of 5.7 is the distal histidine (8). It is now known that the pK a of the distal histidine is less than 4.7 in metmyoglobin (12). In general, the Alberty group did not investigate the reactions to high enough pH to evaluate the rate of the reaction between azide and hydroxymetmyoglobin. However, Ver Ploeg et al. (25, 26) determined the rate of cyanide binding to sperm whale metmyoglobin between pH 4.7 and 10.6 and found that ionization of the heme-bound water in metmyoglobin, with a pK a of 8.9, substantially decreased the rate of cyanide binding. We have completed studies of the binding of imidazole, 1-methyl imidazole (27), and fluoride (21) to horse metmyoglobin over extended pH ranges. Fluoride binding has been investigated between pH 3.4 and 11, while the imidazole studies spanned the pH range, 4.2 to 11.5. We found that the association rate constants were influenced by three different heme-linked ionizations. Ionization of the heme-bound water, with pK a 8.9, decreases the rate of binding of all ligands. A second protein group, with a pK a value of 6.2, appears to influence the binding of the imidazolium cation. This group is probably the same as the pK a 5.7 group found

METMYOGLOBIN/AZIDE KINETICS

by Alberty and co-workers in sperm whale metmyoglobin. Although Alberty suggested that this group is the distal histidine, we argue that it is His-97 (27). His-97 is in the proximal heme pocket and it is thought that placing a positive charge on His-97 through protonation decreases the binding rate of positively charged ligands such as the imidazolium cations. Protonation of His-97 appears to have little influence on the binding of neutral ligands such as imidazole and 1-methyl imidazole (27). This interpretation is consistent with the observation that the group with a pK a of 6.2 has no effect on the binding of neutral hydrofluoric acid (21). However, since hydrofluoric acid binds to metmyoglobin significantly faster than the imidazole species, we were able to investigate HF binding to pH 3.4. We found a third ionization, with a pK a of 4.4 6 0.5, which increases the rate of hydrofluoric acid binding threefold. We believe that this group is the distal histidine, His-64 (21). The dissociation rate constant is also dependent upon pH (Fig. 3) and appears to be influenced by a single ionizable group in the complex. We believe that the ionizable group in the metmyoglobin/azide complex that influences the dissociation rate constant is the distal histidine. The crystallographic structure of metmyoglobin/azide indicates a hydrogen bond between the distal histidine and bound azide (1, 19). The binding of hydrazoic acid to metmyoglobin is similar to the binding of hydrofluoric acid (21). Ionization of both the distal histidine and the heme-bound water influence the rate of azide binding while ionization of the group with pK a of 6.2 has no effect the reaction. The simplest kinetic mechanism we have found that fits the pH dependence of the association and dissociation rate constants is shown symbolically in Scheme 1. Scheme 2 gives a structural interpretation to the kinetic mechanism in Scheme 1. Scheme 1 is part of a more complex kinetic mechanism we have previously proposed to explain the binding of neutral, anionic, and cationic species to metmyoglobin (27). The complete mechanism involves three ionizable groups with apparent pK a’s designated pK P1, pK P2, and pK P3. These three ionizable groups are attributed to His-64, His-97, and the heme-bound water, respectively. Three ionizations generate four different protonated states of metmyoglobin which can have differential reactivities toward ligands (27). The rate constants for interaction between a neutral ligand and the four protonated states of metmyoglobin are designated k 1 , k 2 , k 3 , and k 4 , for the most protonated to least protonated state of metmyoglobin, respectively. If a cationic ligand binds, the rate constants are distinguished with a prime symbol and if an anionic ligand binds, the rate constants are designated with a double prime symbol. In Scheme 1, we allow for the direct binding of both HN 3 and N 32. His-97 does not influence the observed

153

SCHEME 1. Minimal mechanism necessary to explain the pH dependence of azide binding to metmyoglobin. Both hydrazoic acid (HN 3) and the azide anion (N 32) can diffuse into the heme pocket and react with the heme iron. Two ionizable groups on the enzyme influence the rate of binding. pK P1 is assigned to the distal histidine, His-64. HP represents metmyoglobin with His-64 protonated and P represents metmyoglobin with His-64 unprotonated. pK P3 is the ionization of the heme-bound water, producing hydroxymetmyoglobin (POH). An additional ionizable group in the protein, designated by pK P2, was previously found to influence the binding of imidazole and 1-methylimidazole to metmyoglobin (27). However, this group, which is believed to be His-97, does not influence the binding of azide and is not included in this scheme. Likewise, in the original mechanism, k 2 and k 3 represented the rates of ligand binding to metmyoglobin when His-97 was protonated and unprotonated, respectively. In this mechanism the rate constants k 23 and k023 are used to represent HN 3 and N 32 binding to metmyoglobin irrespective of the state of protonation of His-97.

binding of azide to metmyoglobin and its ionization, designated by pK P2, is not indicated in Scheme 1. HP represents the protein with the distal histidine proton-

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SCHEME 2. Structural interpretation of the kinetic mechanism presented in Scheme 1.

ated (see Scheme 2). Both HN 3 and N 32 can react with HP. When HN 3 diffuses into the heme pocket and displaces the heme-bound water (Scheme 2), a hydronium ion dissociates from the complex and the azide anion binds to the heme iron, k 1 . When N 32 diffuses into the heme pocket of HP, it displaces the hemebound water directly, k 01 . In both cases, the bound azide anion is stabilized by hydrogen-bonding to the protonated distal histidine (Scheme 2), giving rise to the azide/metmyoglobin complex designated HPL in Scheme 1. Metmyoglobin with the distal histidine unprotonated is represented by P in Scheme 1. HN 3 binds to P

with a rate constant designated k 23 . 3 In this reaction the proton of HN 3 is transferred to the distal histidine and retained in the complex (HPL) at low pH. When the azide anion reacts with P, k 023 , the azide anion displaces the heme-bound water and forms the complex designated PL in Scheme 1. In the metmyoglobin/ 3 In the original mechanism (27) k 2 and k 3 represent the rate of ligand binding to metmyoglobin when His-97 is protonated and unprotonated, respectively. Protonation of His-97 has no influence on the binding of HN 3 so that k 2 and k 3 are equal. In Scheme 1, protonation of His-97 is not explicitly shown and k 2 and k 3 of the original mechanism are represented by k 23 in Scheme 1.

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azide complex, the proton bound to the distal histidine dissociates with an apparent pK a designated pK C1 in Schemes 1 and 2. Both HN 3 and N 32 can bind directly to the alkaline form of metmyoglobin, POH, displacing a water, k 4 , or hydroxide ion, k 04 . pH dependence of k a and k d for the azide/metmyoglobin complex. The apparent association and dissociation rate constants derived from Scheme 1 are given by Eqs. [5] and [6]. k1

ka 5

S S

D D DS D S D D

@H 1 # KL 1 k 23 1 k 01 K P1 K P1 K P3 KL K P3K L 1 k4 1 k 02 3 1 k 04 KL @H 1 # @H 1 # 2 K P3 @H 1 # KL 111 11 K P1 @H 1 # @H 1 #

S

@H 1 # K C1 1 ~k 24 1 k 02 23 1 k 02 4 @OH 2 #! @H 1 # 11 HC1

[5]

~k 21@H 1 # 1 k 223 1 k 02 1 !

S

kd 5

[6]

Since k d appears to reach plateau values at high and low pH (Fig. 3), the bimolecular rate constants involving the hydrogen and hydroxide ions in Eq. [6] must be negligible. Eliminating the k 21 and k 024 terms in Eq. [6] gives Eq. [7]. @H 1 # 1 ~k 24 1 k 02 23 ! K C1 @H 1 # 11 K C1

~k 223 1 k 02 1 ! kd 5

[7]

The data shown in Fig. 3 were fit to Eqs. [5] and [7] using nonlinear least-squares regression. The best-fit values are given in Table II. The solid lines in Fig. 3 were calculated using the best-fit values for the parameters. Scheme 1 fits the pH dependence of both k a and k d within experimental error and the mechanism satisfies microscopic reversibility. Evaluation of the rate constants in Scheme 1. From fitting the pH dependence of k a to Scheme 1, we can determine the values of k 1 and k 04 uniquely. In theory, we can only determine the values for combinations of the four remaining rate constants, (k 23 1 k 01 K L /K P1) and (k 4 K P3/K L 1 k 023 ) (Table II). However, based on previous studies which show that all ligands bind more slowly to hydroxymetmyoglobin than to aquometmyoglobin, we know that k 4 has to be less than k 23 and k 23 has a maximum value of 3.8 3 10 5 M 21 s 21 (Table II). With k 4 less than 3.8 3 10 5 M 21 s 21, then k 4 K P3/K L is

TABLE II

Kinetic Parameters for Azide Binding to Horse Metmyoglobin a Parameter k1 KL k 23 1 k 01 K P1 K P3 k4 1 k 02 3 KL k 04 pK P1 pK P3 c pK L c

Value

Parameter

Value

(2.5 6 0.8) 3 10 6

k 21

Nb

(3.8 6 1.0) 3 10 5

k 223 1 k 021

18 6 6

(4.7 6 0.3) 3 10 3 5.7 6 3.2 4.0 6 0.3 8.88 4.54

k 24 1 k 0223 k 024 pK C1

0.16 6 0.02 Nb 4.4 6 0.2

a Experimental conditions: 0.1 M ionic strength, 25°C. Parameters are defined in Scheme 1 of the text. Bimolecular and unimolecular rate constants are in units of M 21 s 21 and s 21, respectively. b Makes negligible contribution to the observed dissociation rate constant. c pK P3 and pK L fixed at experimental values.

less than 17 M 21 s 21, making an insignificant contribution to (k 4 K P3/K L 1 k 023 ) (Table II). The value of k 023 is equal to (4.7 6 0.3) 3 10 3 M 21 s 21. Knowing the value of k 023 we can estimate the value of k 01 . The rate constants k 01 and k 023 represent the reaction of N 32 with aquometmyoglobin when the distal histidine is protonated and unprotonated, respectively. It is now thought that protonation of His-64 is coupled to a conformational change in the protein between “open” and “closed” forms (20). In the closed conformation, His-64 is buried within the heme pocket and there is no direct access for ligands between the solvent and the distal heme pocket. This is the structure observed in the crystal structure of metmyoglobin (28). When His-64 protonates, the imidazole side chain moves out of the heme pocket into the solvent, opening a channel which facilitates ligand entry into the distal heme pocket (20). The rates of fluoride binding to metmyoglobin (21) and CO binding to myoglobin (29) increase by a factors of 3 and 7, respectively, when His-64 is protonated. If we assume that the rate of N 32 binding increases by as much as 10-fold upon His-64 protonation, k 01 could be as large as 4.7 3 10 4 M 21 s 21. If k 01 were 4.7 3 10 4 M 21 s 21, it would still make a negligible contribution to the parameter (k 23 1 k 01 K L /K P1) (Table II), and k 23 is equal to (3.8 6 1.0) 3 10 5 M 21 s 21. These considerations indicate that the rate of HN 3 binding increases by a factor of ;7 upon protonation of His-64, consistent with the CO and fluoride binding data. Using the above estimates for the rate constants, one can show that the binding of HN 3 is responsible for the observed rate below pH 6.5 and that binding of N 32 to aquometmyoglobin, k 023 , makes the dominant contribution to k a between pH 6.5 and 11.5.

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Microscopic reversibility arguments can be used to estimate the values of k 223 , k 24 , k 021 , and k 0223 (Table II). The thermodynamic cycle involving the binding of HL is Scheme 1 requires that k 24 5 k 223

k 4 K P3 . k 23 K C1

[8]

On the right-hand side of Eq. [8], the maximum value of k 223 is 18 s 21 (Table II); the ratio of k 4 /k 23 must be less than 1 since all known ligands bind to hydroxymetmyoglobin more slowly than to aquometmyoglobin, and the values of K P3 and K C1 are known. Combining these facts leads to a value of k 24 less than 6 3 10 24 s 21. If k 24 is less than 6 3 10 24 s 21 then k 0223 equals 0.16 s 21 (Table II). Likewise, the thermodynamic cycle involving binding of L 2 in Scheme 1 requires that k 02 1 5 k 02 23

k 01 K C1 . k 02 3 K P1

[9]

The values of k 0223 , K C1, and K P1 are known. The ratio of k 01 /k 023 will be larger than unity. The rate constants k 01 and k 023 represent the binding of N 32 to metmyoglobin when His-64 is protonated and unprotonated, respectively. From arguments presented above, myoglobin binds ligands 3 to 7 times more rapidly when His-64 is protonated due to stabilization of a conformation with an open channel to the distal heme pocket. If we assume that N 32 binds up to 10 times faster to metmyoglobin when His-64 is protonated, then k 021 # 0.6 s 21, contributing less than 4% to the term (k 223 1 k 021 ) in Eq. [7] (Table II). Consequently, k 223 equals 18 s 21 and Eq. [7] can be simplified to Eq. [10]. @H 1 # 1 k 02 23 K C1 kd 5 [10] @H 1 # 11 K C1 The effect of ionic strength on k a. We have found that primary kinetic salt effects are very small in the binding of imidazole and fluoride to metmyoglobin (21, 27). In both cases binding of neutral species make the dominant contributions to the observed association rate constant. In general, inclusion of secondary kinetic salt effects, the effect of ionic strength on the pK a of groups involved in the reaction, are sufficient to account for the ionic strength dependence of imidazole and fluoride binding to metmyoglobin. Our analysis of the pH dependence of the binding of azide to metmyoglobin based on the mechanism shown is Scheme 1 indicates that N 32 binding dominates the observed rate k 223

constant above pH 6.5. We would expect to observe a significant primary kinetic salt effect on k a between pH 7 and 10 (Fig. 3). The apparent bimolecular association constant, k a, for binding of azide to metmyoglobin is a complex expression involving four rate constants (k 1 , k 23 , k 023 , and k 04 ) and three acid dissociation constants (K P1, K P3, and K L ) (Eq. [5]). The ionic strength dependence of two of the acid dissociation constants (K L and K P3) is known from the experiment (Fig. 6) (23, 27, 30). We initially tried to determine if secondary kinetic salt effects could account for the observed ionic strength dependence of k a by simultaneously fitting both the pH (Fig. 3) and ionic strength (Fig. 4) dependence of the observed rate constant. During the fitting procedure, expressions describing the ionic strength dependence of K P3 (27) and K L (Eq. [4]) were substituted into Eq. [5]. We assumed that the ionic strength dependence of pK P1 could be described by

pK P1 5 pK 0P1 1 S P1

ÎI , ~1 1 ÎI!

[11]

with pK°P1 and S P1 used as adjustable parameters. We also assumed that the four rate constants (k 1 , k 23 , k 023 , and k 04 ) were independent of ionic strength and used their ionic strength-independent values as adjustable parameters, giving a total of six adjustable parameters to simultaneously fit the pH and ionic strength dependence of k a. We were not able to fit the ionic strength dependence of k a based upon these assumptions. Since secondary kinetic salt effects were insufficient to account for the ionic strength of the association reaction, we included primary kinetic salt effects for the reactions involving N 32, k 023 , and k 04 . We assume that the ionic strength dependence of the rate constants for the reactions involving azide anion can be described by

log k 0i 5 log k 0°i 1 S i

ÎI , ~1 1 ÎI!

[12]

where log k 0°i and S i are adjustable parameters. This fitting procedure requires eight adjustable parameters, and a satisfactory fit to both the pH and ionic strength dependence of k a was obtained. The solid lines in Fig. 4 were calculated based on the best-fit parameters determined in this analysis. The best-fit values for the parameters are pK °P1 5 3.6 6 0.3, S P1 5 1.9 6 0.7, k 1 5 (2.0 6 0.2) 3 10 6 M 21 s 21, k 23 5 (4.7 6 0.3) 3 10 5 M 21 s 21, k 0°23 5 (6.6 6 0.2) 3 10 3 M 21 s 21, S 23 5 20.97 6 0.10, k 0°4 5 1.2 6 0.8 M 21 s 21, and S 4 5 14.3 6 0.8.

METMYOGLOBIN/AZIDE KINETICS

The one surprise in simultaneously fitting the pH and ionic strength dependence of k a is that the ionic strength dependence of k 023 is anomalous. The slope of Eq. [9] for k 023 , S 23 , gives a best-fit value of 20.97 6 0.10, indicating that k 023 decreases with increasing ionic strength. The isoelectric point of metmyoglobin is 7.2 (31) and the net charge on metmyoglobin is negative over most of the pH region where k 023 dominates the observed rate of binding, pH 6.5 and 11.5. In general, the rates of reactions between ions of like charge increase with increasing ionic strength, contrary to the results obtained here. This may indicate that azide anion accesses the heme pocket through a region of the protein which has a localized positive charge or it may indicate a short-coming of the analysis. It may be too optimistic to assume that a single value of S 23 can describe the ionic strength dependence of k 023 over a pH region where the net charge on metmyoglobin changes from 12 to 218 (31). We can conclude, however, that while secondary kinetic salt effects are sufficient to describe the ionic strength dependence of the association reaction for imidazole (27) and fluoride (21) binding, secondary kinetic salt effects cannot account for the ionic strength dependence of azide binding. We believe that this is strong evidence for the correctness of Scheme 1 and that both HN 3 and N 32 contribute to the observed rate of binding over appropriate pH ranges. The effect of ionic strength on k d. According to Eq. [10], only k 223 , k 0223 , and K C1 influence the dissociation rate between pH 4.75 and 11. The empirical expression for the ionic strength dependence of pK C1 given in Eq. [13] is used in the simultaneous fit of the pH and ionic strength dependence of k d. pK C1 5 pK 0C1 1 S C1

ÎI

~1 1 ÎI!

[13]

Four adjustable parameters are used to simultaneously fit the pH and ionic strength dependence of log k d. These four parameters are the values of k 223 and k 0223 at zero ionic strength, the value of pK C1 at zero ionic strength, and S C1. The best-fit values for k 223 and k 0223 at zero ionic strength are 17 6 3 and 0.20 6 0.01 s 21, respectively; pK°C1 is 4.1 6 0.1; and S C1 is 1.9 6 0.2. The calculated secondary kinetic salt effects are shown in Fig. 5 by the solid lines. The fit is excellent and the ionic strength dependence of pK C1 is quite reasonable. In contrast to the ionization of the distal histidine in the free enzyme, which has a relatively small effect on the association rate constant (factor of seven), protonation of the distal histidine in the azidometmyoglobin complex increases the dissociation rate by two orders of magnitude. Note also that the ionic strength dependence of pK C1 and pK P1 are identical with best-fit val-

157

ues of 1.9 6 0.7 and 1.9 6 0.2 for S P1 and S C1, respectively. CONCLUSIONS

We have investigated the association rates of imidazole (27), 1-methylimidazole (27), fluoride (21), and azide (this study) to horse metmyoglobin over an extended range of pH. Metmyoglobin discriminates between the charged and neutral forms of these ligands, with the neutral form of the ligand binding more rapidly than the charged form, whether cationic or anionic. Protonation of the distal histidine increases the rate of binding for the neutral forms of the ligands such as HF and HN 3 by factors of 3 and 7, respectively, similar to the enhancement of CO binding rate of myoglobin (20). The pK a for the protonated form of His-64 is 4.2 6 0.3, averaging the values obtained from the binding of fluoride (21) and azide (this study). The small increase in the rate of HF and HN 3 binding upon His-64 protonation is consistent with the dual role of His-64 in ligand binding reactions. Studies with mutant myoglobins indicate that His-64 controls access to the distal heme pocket via a gating mechanism and that an open gate conformation can increase the rate of azide anion binding 10 3-fold (18). Protonation of His-64 stabilizes the open conformation (20) but only a 7-fold enhancement of the HN 3 binding rate is observed upon His-64 protonation. This suggests that the unprotonated form of His-64 facilitates azide binding by base catalysis, aiding in the removal of the proton from HN 3 within the distal heme pocket. These two roles of the distal histidine, gating and base catalysis, compensate, and protonation of His-64 has a relatively small effect on the rate of HN 3 binding. Ionization of the heme-bound water, with a pK a of 8.9, decreases the rate of ligand binding. In the case of neutral imidazole and 1-methylimidazole the rate decreases by factors of 6 and 3, respectively. The rates of HF and HN 3 binding to hydroxymetmyoglobin do not contribute to the observed rate of complex formation. The rate of binding of imidazole, HF, and HN 3 to aquometmyoglobin with the distal histidine unprotonated are 3.5 3 10 2, 4.7 3 10 4, and 3.8 3 10 5 M 21 s 21, respectively. There is a more stringent discrimination between neutral and anionic forms of the azide and fluoride than there is between the neutral and cationic forms of imidazole by aquometmyoglobin, probably due to charge-density effects. The imidazolium cation binds with a rate 34% smaller than imidazole, while the azide and fluoride anions bind 80 and 840,000 times more slowly than their respective weak acids. Conversion of aquometmyoglobin to hydroxymetmyoglobin causes an additional decrease of 300- to 800-fold in the rate of fluoride and azide anion binding, respectively.

158

LIN ET AL.

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