THE JOURNAL OF BIOLOCKAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 6, Issue of February 25, pp. 3X&3176,1990 Printed in U.S. A.

The Effects of Amino Acid Substitution at Position E7 (Residue 64) on the Kinetics of Ligand Binding to Sperm Whale Myoglobin*

(Received for publication, August 23, 1989)

Ronald J. RohlfsS, Antony J. Mathews, Theodore E. Carver& and John S. Olson$ From the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251

Barry A. Springer, Karen D. Egeberg, and Stephen G. Sligarll From the Departments of Chemistry and Biochemistry, University of Zllinois, Urbana, Illinois 61801

Association and dissociation rate constants were measured for 02, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglo- bins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8961-8965). The Hise4 + Gly substitution produced a sterically unhindered myo- globin that exhibited ligand binding parameters simi- lar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Glya4 (-10’ M-’ s-‘) > Vale4 = Leua4 (-10’ M-’ s-l) > Mete4 > Phea4 = Hise4 = Glne4 ( lo’-lo3 M-’ s-‘) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimo- lecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the Hise4 + Arg and Hisa + Asp mutants were abnormally high (l-5 X 10’ M-l s-l), suggesting that Arge4 and Aspe4 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the associ- ation rate constants for 02 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except Hisa + Gln ap- proached or were larger than those for chelated model heme (i.e. -1 X 10’ M-’ s-’ for O2 and -1 X 10’ M-’ s-l for CO), whereas the corresponding rate parameters for myoglobin containing either Glna4 or Hisa were 5- to lo-fold smaller. This result suggests that a major kinetic barrier for 02 and CO binding to native myo- globin may involve disruption of polar interactions between Hise4 and water molecules found in the distal pocket of deoxymyoglobin. Finally, the rate and equi- librium parameters for O2 and CO binding to the Hisa + Gln, Hise4 -) Val, and Hise4 -, Leu mutants were compared to those reported previously for Asian ele- phant myoglobin (Gln-E7), Aplysia limacina myoglo-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipients of graduate fellowships from National Institute of Medical Science Training Grant GM-07933.

$ Supported by United States Public Health Service Grant GM- 35649, Grant C-612 from the Robert A. Welch Foundation, and Grant 4073 from the Advanced Technology Program of the Texas Higher Education Coordinating Board.

7 Supported by United States Public Health Service Grants GM- 33775 and GM-31756.

bin (Val-E7), and monomeric Hb II from Glycera di- branchiuta (Leu-E7).

The distal histidine in mammalian myoglobins (residue 64) is located at the E7 helical position.’ One edge of the imidazole side chain is directly adjacent to bound ligand, and the other is facing outward toward the surface of the protein (Phillips, 1980; Shaanan, 1983). This orientation is thought to promote stabilization of the iron-O2 complex by hydrogen bonding between the second oxygen atom and the t-amino nitrogen of Hi@“. Direct evidence for the existence of this interaction was obtained by Phillips and Schoenborn (1981) in neutron dif- fraction studies with sperm whale myoglobin. The close prox- imity of Hi@* required for hydrogen bonding favors a bent geometry for the bound ligand over a linear orientation. Thus, the distal histidine is thought to reduce the affinity of myo- globin for CO and alkyl isocyanides by pushing the bound ligand off an axis perpendicular to the heme plane (Collman et al., 1976). Kuriyan et al. (1986) have shown that in sperm whale myoglobin His64 is slightly displaced by bound CO which exhibits two bent conformations. Johnson et al. (1989) observed a much larger displacement of the distal histidine in the high resolution crystal structure of ethyl isocyanide sperm whale myoglobin, and the bound isocyanide was markedly distorted from a linear geometry. In this latter complex, about 40% of the E7 imidazole side chains were rotated about the C,-CB bond to create a direct channel from the solvent to the heme iron atom. This observation suggests that Hisa may act as a “gate” for ligand entry into the distal pocket, in agreement with predictions based on dynamics calculations (Case and Karplus, 1979; Kottalam and Case, 1988) and with earlier x- ray studies of metmyoglobin complexes containing large bound ligands (Ringe et al., 1984; Bolognesi et al., 1982). Taken together, these crystallographic studies provide evi- dence that HisM plays an important role in determining the rate and extent of ligand binding to myoglobin. However, a key problem is how to determine experimentally the func- tional and therefore physiological significance of the observed structural interactions.

Three approaches have been used to assess the importance of distal protein-ligand interactions in myoglobins and he- moglobins. The first approach is to compare the rate and equilibrium parameters for the binding of ligands which differ in size and chemical properties. The iron-02 complex is bent

‘The alphanumeric code (e.g. E7) refers to the position of the residue within the helices and loops of myoglobin and hemoglobin (Dickerson and Geis, 1983). In the case of native sperm whale myoglobin, E7 corresponds to position 64 in the amino acid sequence.

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Ligand Binding to Mb E7 Mutants

and stabilized by polar environments since it has a partial negative charge on the bound oxygen atoms; in contrast, the iron-CO bond is linear and affected less specifically by solvent conditions since it is apolar (Traylor et al., 1985, a and b; Suslick et al., 1984). Alkyl isocyanides form a homologous series of ligands which can be used to examine steric inter- actions in the distal pockets of hemeproteins (Mims et al., 1983). The second approach is to synthesize model heme compounds that contain distal features thought to be impor- tant in ligand binding and then to compare the rate parame- ters of these synthetic active sites to those of myoglobin and hemoglobin (Traylor et al., 1979; Collman et al., 1983). The third and most direct approach is to compare the ligand binding properties of myoglobins containing different amino acids at the E7 position. In the past, these types of studies were limited to naturally occurring substitutions such as Asian elephant myoglobin (Gln-E7), Aplysiu limucinu myoglo- bin (Val-E7), and monomeric HbII from Glyceru dibranchiutu (Leu-E7) (e.g. see Mims et al., 1983).

The method of choice to resolve the functional contribu- tions of Hiss4 is to carry out site-directed mutagenesis at this position. Nagai and co-workers (Nagai and Thtigersen, 1987) have used a fusion protein expression system in Escherichiu coli to produce mutations at the E7 position in the cy and fi chains of human hemoglobin, and Mathews et al. (1989) have presented comparisons of the rates of Os, CO, and methyl isocyanide binding to the R-state forms of these proteins. Springer and Sligar (1987) have constructed a synthetic gene for sperm whale myoglobin which is expressed as the fully functional holoprotein in E. coli. Springer and Sligar’s expres- sion system was used to produce synthetic myoglobins con- taining His(wild type), Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp at residue 64. Adopting the approach of Mims et al. (1983), we measured the rates of Os, CO, and alkyl isocyanide binding to all nine genetically engineered proteins and native myoglobin by rapid mixing and flash photolysis techniques and compared the results to those for unhindered chelated protoheme. Our purpose was 2-fold. We wanted to assess the importance of the size, stereochemistry, charge, and polarity of the amino acid side chain at position E7 on the physiolog- ical properties of myoglobin and to examine this set of mu- tations in order to choose which proteins have unusual enough functional properties to warrant more detailed biophysical and structural studies.

In two previous short communications, we compared the effects of the His-E7 ---* Gly substitution on the parameters for OS, CO, and methyl isocyanide binding to sperm whale myoglobin and the (Y and fi subunits of R-state human he- moglobin (Olson et al., 1988) and surveyed the autooxidation properties and CO and O2 equilibrium constants of the H64G, H64V, H64M, H64F, H64R, and H64D mutants’ (Springer et al., 1989). The current work complements and extends these earlier studies by including kinetic measurements of the bind- ing of six alkyl isocyanides, focusing on the kinetic mechanism of ligand binding, and presenting data for 02, CO, and iso- cyanide binding to two new and key E7 mutants, H64Q and H64L.

’ The amino acid at the distal histidine position in the site-directed mutants is referred to as 64 for simple comparison with the native protein even though the E. coli proteins contain an additional amino acid at the N terminus (i.e. GlyG4 myoglobin). The mutations are listed in the text using the single amino acid abbreviations (i.e. the Hir?’ + Gly, His?’ -+‘val, His’ + Leu, HisG4 + Met, Hi@“ + Phe, His? + Gln, HirY + Arg, and His” + Asp substitutions are desig- nated as the H64G, H64V, H64L, H64M, H64F, H64Q, H64R, and H64D mutations, respectively).

MATERIALS AND METHODS

Wild-type and mutant sperm whale myoglobins were expressed in E. coli from a synthetic gene and purified to homogeneity as described by Springer and Sligar (1987) and Springer et al. (1989). Native sperm whale myoglobin (Type II) was obtained from Sigma, stored at -20 “C, and used without further purification. Isocyanides were synthesized, and stock solutions were prepared as described by Reisberg and Olson (1980a). Stock solutions of oxygen, carbon monoxide, and nitric oxide were prepared by equilibrating 0.1 M phosphate buffer, pH 7.0, with 1 atm of the pure gas.

Rapid mixing experiments were carried out in a Gibson-Dionex stopped-flow apparatus equipped with an On-Line-Instruments-Sys- tems (OLIS) model 3820 data collection system. For association experiments, concentrated metmyoglobin samples were diluted in deoxygenated, 0.1 M potassium phosphate buffer, pH 7.0, to a concen- tration of Z-10 pM, reduced with sodium dithionite, and mixed with various concentrations of CO and isocyanides under pseudo-first order conditions at 20 “C. Dissociation reactions were also carried out in the stopped-flow apparatus. For these experiments, a 2-5 pM heme solution of the liganded complex was mixed with a high concentration of displacing ligand as described under “Results.”

Association time courses for CO and alkyl isocyanide binding were also determined by conventional flash photolysis using an apparatus consisting of two photographic strobe units (Sunpak 544) equipped with thvristor ouenching devices (Reisberg and Olson. 1980b). The flash was set to a rectangular pulse which-was -0.5 ms in duration with rise and decay times ~0.1 ms. Recombination after the flash was monitored as transmittance voltages that were collected and con- verted to absorbance changes with a high speed A/D converter and OLIS, Inc. software. Stoppered l-cm path length fluorescence cu- vettes containing a few grains of solid dithionite were flushed with NP. Anaerobic solutions containing 5 pM metmyoglobin and known concentrations of carbon monoxide or isocyanide were injected into the cells. Soret and visible absorption spectra were recorded after each flash experiment.

Oxygen association rate constants were measured by flash photo- lysis using a pulsed dye laser system (Phase-R Model 2100B) which allowed the measurement of traces exhibiting half-times in the range of 1Om6 to lo-” s (see Morris and Sawicki, 1981). The rapid rates of autooxidation of ferrous iron for all of the E7 mutants except H64Q required a more complex method of sample preparation. A small Sephadex G-25 column (0.25 X 5 cm) was equilibrated with 0.1 M phosphate buffer, pH 7.0, saturated with 1 atm of 0,. Metmyoglobin samples were reduced with a small amount of solid dithionite and immediately passed through the column. The resultant Mb02 sample was eluted directly into a stoppered, l-mm path length cuvette previously flushed with 1 atm of pure 0,. The sample was diluted to a final heme concentration of 50 FM with phosphate buffer equili- brated with 1 atm of 0,. The sealed cuvette was then brought to 20 “C by partial submersion in a water bath for -60 s and placed in a water- jacketed cell holder. The photolysis and monitoring beams were focused on the front face of the cell, and voltage transmittance signals were collected on a digitizing oscilloscope (Tektronix model 2430) and then transferred to an IBM PC-AT computer for analysis.

RESULTS

Determination of Association and Dissociation Rate Con- stunts-Sample time courses for ligand binding to deoxymy- oglobin mutants are shown in Fig. lA. These traces were obtained from stopped-flow rapid mixing experiments in which 5 I.~M protein was reacted with 50 FM methyl isocyanide. Under these conditions, the reactions for native and E. coli wild-type myoglobin exhibited half-times equal to -70 ms, whereas that for the H64G mutant was extremely small, -1.2 ms, so that most of the time course was lost in the dead time of the stopped-flow apparatus (-3 ms). In order to determine the association rate constant for the H64G mutant, rapid mixing experiments were carried out at lower ligand concen- trations (525 PM), and conventional flash photolysis tech- niques were used to increase the time resolution of the meas- urements to half times 20.1 ms. Photolysis experiments were also carried out to compare the association kinetics of myo- globin samples that had been deoxygenated for -5-10 min

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Ligand Binding to Mb E7 Mutants

FIG. 1. A, observed time courses for methyl isocyanide binding to E7 myoglobin mutants. The conditions after mixing for all reactions were 5 fiM heme, 50 pM methyl isocyanide, 0.1 M potassium phosphate, pH 7.0, 20°C. The time courses were monitored at 431 nm. Two hundred points were collected and analyzed for each time course, but only every fourth point is shown in the figure to allow visualization of the fitted curve. The solid lines represent single exponential fits to the complete data sets. The fraction deoxymyoglobin was calculated by dividing the observed A absorbances at each time by the fitted total absorbance change which takes into account the dead time of the stopped-flow apparatus (3 ms). Symbols: open triangles, Gly6’ myoglobin (Mb)&+. = 593 s-l; open squares, ValM Mb, &bs = 44 s-i; closed triangles, PheG4 Mb, kobs = 11 s-l; closed circles, Arg64 Mb, kobs = 53 s-l; open diamonds, Meta Mb, kobs = 15 s-i; open circles, His%- E. coli Mb, ko,,a = 8 s-l; closed squares, HisG4-native Mb, &,bs = 8 s-i. B, observed time courses for the reaction of CO with the O2 complexes of E7 myoglobin mutants. The conditions after mixing were 5 pM heme, 737.5 pM 02, 50.5 pM CO, 0.1 M potassium phosphate, pH 7.0, 20 “C. All reactions were monitored at 424 nm. The symbols are the same as described for panel A and represent every fourth time point from the original data file. The solid lines represent single exponential fits to the time courses: r&s (GlyG4) = 5 s-‘; r0ba (Vale4) = 44 s-i; r&,‘,ba (PheG4) = 41 s-‘; robs (Arge4) = 4 s-l, r&s (Metc4) = 7 s-l; robs (HiP-E. coli) = 0.05 s-i; robs (Hise4-native) = 0.05 s-i. The MetG4 data were also fitted to a two-exponential expression yielding: robs (fast) = 26 s-‘, 60% of the total absorbance change at 424 nm; robs (slow) = 3 s-i, 40% of the total absorbance change.

prior to reaction with those of deoxymyoglobin generated in 0.5 ms by photolysis to ensure that there were no slow, functionally significant conformational changes accompany- ing ligand removal3 The rates and time courses obtained by rapid mixing and flash photolysis were in agreement for all of the E7 mutants reported in Tables I-III.

Sets of rapid mixing and flash photolysis traces were re- corded at several different CO and alkyl isocyanide concen- trations for all of the mutant myoglobins reported in Tables

3 We have observed differences between time courses measured by rapid mixing and those measured by flash photolysis for CO and alkyl isocyanide binding to the double mutants H64G-V68A and H64G- V68F of sperm whale myoglobin (K. D. Egeberg, B. A. Springer, S. G. Sligar, T. E. Carver, R. J. Rohlfs, and J. S. Olson, unpublished results).

600 Mb + MNC so0 tGlY

2

3 400 “a1

2 t 300

B : 200 Phc .

I k._:::i His

0 loo A- h

0 0 100 200 300 400 500

[MNCI micromolar

FIG. 2. Dependence of the observed association rate on li- gand concentration for several E7 mutants of sperm whale myoglobin at pH 7.0, 20%. Reactions were carried out in the stopped-flow apparatus as described in Fig. 1 or by conventional flash photolysis techniques as described under “Materials and Methods.” kobs (s-i) is plotted uersus the concentration of methyl isocyanide (MNC) after mixing or in the flash cuvette. The solid lines represent fits to Equation 1 with the parameters listed in Table I. Symbols: solid triangles, GlyG4 myoglobin (Mb); solid squares, Va16* Mb; open triangles, PheG4 Mb; solid circles, Hisa (native) Mb.

I-III. In each case, simple pseudo-first order kinetic behavior was observed when the ligand was in excess and the time courses were fitted to a single exponential expression using an iterative, nonlinear, least squares algorithm. The depend- ence of the observed rate on ligand concentration was exam- ined using Equation 1:

kobs = k’[Xl + kx (1)

where k’ is the bimolecular association rate constant and k, the dissociation rate constant. Sample plots of Kobs uersus ligand concentration are presented in Fig. 2. A linear depend- ence was observed for every protein-ligand combination ex- amined, including the H64G mutant for which extremely rapid reactions were observed (Figs. IA and 2).

Ligand dissociation was measured directly by carrying out replacement reactions in the stopped-flow apparatus. In these experiments, a myoglobin-ligand complex (MbX) was mixed with a replacing ligand (Y) which exhibited a much greater affinity for the protein and a smaller dissociation rate con- stant. At high replacing ligand concentrations, the observed rate of replacement, r&s, is given by

robs = kA1 + (k~lXllk~lYl)1 (2)

where ki and lz, represent the association and dissociation rate constants, respectively, for X, the ligand bound to the protein before mixing; k$ and ky are the corresponding rate constants for Y, the replacing ligand (Olson, 1981). Oxygen and isocyanide dissociation reactions were measured using CO as the replacing ligand. For CO dissociation reactions, the MbCO complex was mixed with buffer equilibrated with 1 atm of NO gas. Since kho >> k&o for all hemeproteins (Rohlfs et al., 1988), the observed replacement rate constant was directly equal to the CO dissociation rate constant (i.e. k: [X] /k$[ Y] << 1 in Equation 2).

The final values of k’ and k reported for O2 and isocyanide binding in Tables I-III were obtained by fitting sets of ob- served association and replacement rate data simultaneously to Equations 1 and 2. The value of k&o in Equation 2 was determined independently from association experiments with the same stock of mutant protein. For CO reactions, the dissociation rate constant was so small (kc0 = 0.01-0.05 SK’) that the association rate constant was determined from the slope of k’ versus (CO) plots with y intercepts = 0. The association equilibrium constants were calculated from the ratio of the rate constants, K = k’/k.

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Ligand Binding to Mb E7 Mutants 3171

TABLE I Effects of apolar E7 side chains on the rate and equilibrium constants for ligand binding

to sperm whale myoglobin at pH 7.0, 20 “C The errors for native myoglobin were determined as the standard deviation from the mean of at least seven

completely independent determinations with different protein samples over the past 7 years (see Mims et al., 1983, Rohlfs et al., 1988). The average relative error for the rate constants is +lS% and &20% for the equilibrium constants. With the exception of the oxygen data, the E7 mutant rate constants were collected for usually only one protein preparation, and as a result standard deviations could not be computed. The relative errors for native myoglobin are assumed to apply to the mutant parameters (i.e. +20%). The parameter values were rounded off to two significant figures after K was calculated. The isonitrile abbreviations are: MNC, methyl; ENC, ethyl; nPNC, n-propyl; nBNC, n-butyl; iPNC, isopropyl; and tBNC, tert-butyl isocyanide.

Ligand E7 residue (DOSitiOll 64) k’ k K

02

co

MNC

ENC

nPNC

nBNC

iPNC

tBNC

QY Val Leu Met Phe His (native) His (E. coli)

x 10-6 ‘kc’ s-1 140 250”

98 75 75 14 + 3 16 f 3

s-1 1,600

23,000” 4,100” 1,700

10,000 12 f 2 17 -+ 4

x 10-6 M’ 0.088 0.011 0.023 0.045 0.0074 1.2 + 0.3 0.9 + 0.3

GUY 5.8 0.038 150 Val 7.0 0.048 150 Leu 26.0 0.024 1,100 Met 4.6 0.023 200 Phe 4.5 0.054 83 His (native) 0.51 + 0.06 0.019 + 0.005 27 f 8 His (E. coli) 0.50 0.018 28

GUY 10 6.3 1.6 Val 0.71 12 0.059 Leu 1.80 2.1 0.86 Met 0.44 4.0 0.11 Phe 0.18 2.4 0.075 His (native) 0.12 + 0.02 4.3 2 0.3 0.028 + 0.006

His (E. co&) 0.12 4.3 0.028

GUY 15 Val 2.2 Leu 1.0 Met 0.29 Phe 0.093 His (native) 0.069 f 0.01 His (E. coli) 0.074

2.0 4.0 0.15 0.28 0.17 0.30 f 0.03 0.27

7.5 0.55 6.7 1.1 0.56 0.23 + 0.05 0.28

GUY 27 Val 0.76 Leu 0.41 Met 0.13 Phe 0.030 His (native) 0.042 + 0.01 His (E. coli) 0.043

0.4 2.0 0.081 0.17 0.13 0.39 0.33

67 0.38 5.0 0.77 0.22 0.11 0.13

QY 17 0.15 115 Val 0.18 1.6 0.11 Leu 0.25 0.23 1.1 Met 0.082 0.41 0.20 Phe 0.025 0.28 0.089 His (native) 0.030 f 0.002 0.69 + 0.05 0.044 + 0.004 His (E. coli) 0.029 0.60 0.048

GUY Val Met Phe His (native) His (E. coli)

Glr Val Met Phe His (native) His (E. coli)

9.3 0.14 0.035 0.0077 0.012 f 0.002 0.011

5.1 0.0022 0.0039 0.0010 0.0019 + 0.0002

0.9 10 3.9 0.035 0.26 0.13 0.072 0.11 0.53 + 0.01 0.023 + 0.004 0.47 0.024

2.2 0.52 0.29 0.22 0.96 + 0.02 10 0.0016 I.&

2.3 0.0041 0.014 0.0045 0.0020 f 0.0002 0.0013

’ As described in the text, these numbers for 0, association and dissociation have greater uncertainty (-&50%) than those for CO and isonitrile binding.

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Ligand Binding to Mb E7 Mutants

TABLE II Effects of charged and polar E7 amino acids on the rate and

equilibrium constants for ligand binding to sperm whale myoglobin at pH 7.0, 20 “C

Errors and abbreviations are given in Tables I and II. The param- eters for His@ are for native myoglobin.

Ligand E7 residue (position 64) k’

co

MNC

ENC

nPNC

nBNC

iPNC

tBNC

x 10-E M’ s-1

His 14 Gln 24 Arg 79

His 0.51 Gln 0.98 Arg 5.7 ASP 4.4

His 0.12 Gln 0.20 Arg 1.2 ASP 2.9

His 0.069 Gln 0.071 Arg 2.3 ASP 4.5

His 0.042 Gln 0.031 Arg 1.1 ASP 3.0

His 0.030 Gln 0.024 Arg 0.62 Asp 3.3

His 0.012 Arg 0.21 ASP 1.0

His 0.0019 Ax 0.015 ASP 0.14

s-1 x 10-e M’

12 1.2 130 0.18 880 0.090

0.019 27 0.012 82 0.014 400 0.052 85

4.3 0.028 5.6 0.037 5.6 0.21

0.30 0.23 0.15 0.47 0.93 2.5

0.39 0.11 0.12 0.26 0.38 2.8

0.69 0.044 0.31 0.076 0.42 1.5

0.53 0.023 0.61 0.34

0.96 0.0020 0.50 0.030

O2 Complexes of the E7 Mutants-As described by Springer et al. (1989), the H64G, H64V, H64M, H64F, and H64R mutants exhibit autooxidation rates that are -IOO-fold greater than native or wild-type myoglobin. The half-time for the decay of the MbO, complex of these proteins is 5-10 min in 1 atm of 0, at pH 7,20 “C. As a result, substantial amounts

of metmyoglobin had formed in the samples used to determine oxygen association and dissociation rate constants. The ob- served absorbance changes were always less than the maxi- mum expected based on total heme concentration and de- creased rapidly with time after preparation of the oxygenated complex. In the case of the H64D mutant, autooxidation was so rapid that the O2 complex could not be obtained, and only the CO and isocyanide association kinetics of this protein were examined. The H64L and H64Q mutants were not studied by Springer et al. (1989). As expected, the myoglobin containing Le@ autooxidized rapidly, exhibiting a rate simi- lar to that of proteins containing Meta and Phe64. The rate of autooxidation of the protein containing Gln64 was similar to that of native sperm whale myoglobin and the O2 complexes of this mutant were readily prepared and easily studied.

The oxygen association reactions were normally carried out at only one ligand concentration and were further complicated by large bimolecular rates for all of the E7 mutants except H64Q. Reduction of the oxygen concentration was not possi- ble for most of the mutants for two reasons. First, the rate of autooxidation increases with decreasing oxygen concentration in this range, making it more difficult to obtain enough MbO, to carry out the kinetic measurements (Springer et al., 1989), and second, the affinities of some of the mutants are so low that complete saturation is not achieved at oxygen concentra- tions less than 1.0 mM (Tables I-III).

Similar problems occurred for the O2 replacement reactions (Fig. 1B). Normally, in these types of experiments, the [O,]/ [CO] ratio is kept 51.0 so that r&s approaches the true rate constant for O2 dissociation, k, in Equation 2. However, again the high rates of autooxidation dictated that the mutant samples be kept at high O2 concentrations, and since the ko, values were in the range l,OOO-20,000 s-l, the concentration of CO had to be reduced to 5 x 1O-5 M to allow visualization of the time courses in the stopped-flow apparatus. Under these conditions, r& = ko, k&[CO]/k&[Oz] (Equation 2), and the determination of ko, depends on an accurate value for kb,. As described in the previous section, Equations 1 and 2 were solved simultaneously for k& and &,, but, for almost all of the mutants, only single values of kobs and r,,bbbs were used. This limitation, coupled with the small signal changes and very large observed rates, placed greater uncertainty in the rate parameters for oxygen than those for CO and isocyanide binding. However, the equilibrium constants for these mu- tants are well defined by the observed replacement rate con- stants at high [O,]/[CO] ratios since k&o was measured inde- pendently and r&e = k&[CO]/Ko,[Oz].

TABLE III

Comparisons of 0, and CO binding parameters for sperm whale myoglobin, Asian elephant myoglobin, A. lima&a myoglobin, HbII from G. dibranchiata, and synthetic sperm whale myoglobin mutants produced by E. coli

in 0.1 Mpotassium phosphate, pH 7.0, 20 “C The rate constants for Asian elephant myoglobin were taken from Bartnicki et al. (1983); those for Aplysia

myoglobin from Wittenberg et al. (1965 and 1972); and those for monomeric Glycera hemoglobin from Parkhurst et al. (1980) and Rohlfs et al. (1988). Parameter values were rounded off to two significant figures after K and M had been calculated.

Protein 4 b K% k&o ko KC0 M

x 10-6 M-1 s-1 s-1 x 10-B M’ x 10-6 M’ s-1 s-1 x 10-B M’ Kd&, Asian elephant Mb (Gln-E7) 18 18 1.0 0.53 0.0068 78 80 E. coli Mb (GlnE7) 24 130 0.18 0.98 0.012 82 460

Aplysia Mb (Val-E7) 15 70 0.21 0.50 0.020 25 120 E. coli Mb (Val-E7) 250” 23,000” 0.011 7.0 0.048 150 14,000

Glyceru HbII (Leu-E7) 190 1,800 0.11 27 0.042 640 6,000 E. coli Mb (Leu-E7) 98” 4,100” 0.023 26 0.024 1,100 48,000

’ As described in the text, these numbers for 02 association and dissociation have greater uncertainty (-f50%) than those for CO and isonitrile binding.

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Ligand Binding to Mb E7 Mutants

Heterogeneous Kinetic Behavior for H64M-With one ex- ception, the observed time courses for all of the association and replacement reactions studied exhibited simple mono- phasic, exponential behavior. In the case of myoglobin con- taining Met64, heterogeneous time courses were observed for the displacement of all eight ligands in Table I. This phenom- enon was most pronounced for the dissociation of O2 (Fig. 1B) and least pronounced for the dissociation of CO, with the isocyanide replacement reactions exhibiting intermediate be- havior. In contrast, the ligand association time courses for the H64M mutant were monophasic. The biphasic replacement reactions suggest that, in the liganded state, the Met side chain occupies more than one slowly interconverting confor- mation which affects the overall rate of ligand dissociation. In order to simplify comparisons with the other mutants, we chose to represent ligand dissociation from the H64M mutant as a single exponential process. As a result, the dissociation rate constants in Table I for this protein are approximate, particularly in the case of 02.

Properties of Wild-type Myoglobin Expressed in E. coli- One potential criticism of the synthetic sperm whale myoglo- bin used in these and previous studies is the presence of an N-terminal Met. Recently, Phillips et al. (1989) have de- scribed the high resolution (1.9 A) crystal structure of the ferric form of the wild-type sperm whale myoglobin produced by E. coli. Although the extra Met residue does cause the synthetic protein to crystallize in a space group different from that of the native protein, the tertiary structures of the two myoglobins are identical except in the immediate region of the N terminus. The results in Tables I complement these structural observations and show that the detailed functional properties of the ferrous forms of the synthetic and native proteins are also identical. Although the similarity of the 02 and CO binding parameters may not be surprising, the close- ness of the controls for all six alkyl isocyanides (Table II) is an impressive confirmation of structural and functional iden- tity. For example, horse heart and sperm whale myoglobin do exhibit measurably different rate parameters for the binding of some of the larger isocyanides which serve as more sensitive probes of the overall tertiary structure of these proteins (Mims et al., 1983).

DISCUSSION

Production of a Sterically Unhindered Myoglobin--Even in the absence of a high resolution crystal structure, it is clear that the distal pocket of the H64G mutant is sterically un- hindered. The association rate constants for alkyl isocyanide binding to this protein are lOO- to lO,OOO-fold greater than those for native sperm whale myoglobin and show little de- pendence on the length and extent of substitution along the alkyl side chain of the ligand molecule (Table I and Fig. 3). As shown in Fig. 3A, the absolute values of k’ for methyl and ethyl isocyanide binding approach those observed for chelated protoheme suspended in soap micelles. The association equi- librium constants for myoglobin containing Gly6* are also extremely large and very similar to those for chelated proto- heme in soap micelles and for leghemoglobin which has been shown to have an “open” distal pocket (Mims et al., 1983). The linear increase in K with increasing length of the alkyl isocyanide shown in Fig. 3B is presumably due to a hydropho- bic effect involving preferential partitioning of the larger ligands into the unhindered distal pocket of the Gly6* protein. Similar behavior is observed for leghemoglobin (Stetzkowski et al., 1979) and chelated protoheme in soap micelles (Olson et al., 1983).

The results for Glye4 myoglobin support the choice of che-

2” ’ I O’ I” c I’ MNC ENC “PNC “BNC ,PNC ,“NC M Y C E N C q Y N C “ B N C iPNC l”NC

Ligmd Lip”d

FIG. 3. Dependence of the association rate (k’) and equilib- rium (K) constants on ligand size and stereochemistry. A, log(k’) uersus ligand length (n-series) and a-substitution (iso-propyl and tert-butyl); B, log(K) versus ligand length and substitution. Ligand abbreviations are given in Table I. Symbols: open circles, unhindered chelated protoheme in soap micelles (taken from Mims et al., 1983); closed triangles, Glye4 myoglobin (Mb); closed squares, Va16” Mb; solid circles, native Hi? Mb; open trianggles, Phee4 Mb.

lated protoheme as an unhindered standard for quantitating the effects of distal structures on the rate and equilibrium constants for ligand binding to mammalian myoglobins and hemoglobins. These data also support the conclusion of Mims et al. (1983) that steric interaction with the distal histidine is the primary cause of the reduced affinity of native myoglobin for alkyl isocyanides. Finally, the ratio of the equilibrium constants for the native and Glye4 proteins provides an esti- mate of 2.1 to 2.4 kcal/mol for the free energy required to distort the iron-isocyanide complex and move Hise4 partially out of the distal pocket as is observed in the crystal structure of the ethyl isocyanide complex of sperm whale myoglobin (Johnson et al., 1989).

Dependence of the Affinity Constant on the Size and Polarity of the E7 Residue-The CO and O2 equilibrium constants for the H64G, H64V, H64F, H64M, and H64R mutants have been described by Springer et al. (1989). The new results for the H64L and H64Q mutants confirm their basic conclusions. The affinity constant for O2 binding exhibits little dependence on the size of the E7 residue (Tables I and II). The side chain volumes of Val, Leu, Met, and Phe are 75, 102, 104, and 137 ii”, respectively, and those for Asp, Arg, Gln, and His are 58, 113,95, and 101 k, respectively (Creighton, 1983). As shown in Fig. 4, the mutants with apolar amino acid substitutions all exhibit Ko, values 50- to IO@fold less than native myoglo- bin regardless of size. The order of oxygen affinity for the remaining derivatives is His64(native) > Gln’j4 > Gly’j4 = Arg’j4. Ko, for Gly’j4 myoglobin is only lo-fold lower than native, suggesting that solvent water can reach and stabilize the bound O2 (Springer et al., 1989). A similar effect was postu- lated to occur for the H64R mutant since the Arg side chain is thought to point out toward the solvent as in ,B chains (Arg- E7) of Hemoglobin Zurich (Tucker et al., 1979, Springer et al., 1989).

Polarity also appears to be a factor for determining CO association equilibrium constants since all of the E7 mutants exhibited Kc0 values at least 3-fold greater than the native protein. The LeuG4 protein exhibited an abnormally large CO affinity constant (-1 X lo9 M-‘) compared to those for Va164, Met64, and Phe64 myoglobins (all 1-2 X lo8 M-‘; see Fig. 4). This suggests that the stereochemistry at the C4 atom as well as the overall size of the E7 residue plays an important role in determining the extent of CO binding.

The equilibrium constants for isocyanide binding indicate that the size and stereochemistry of the E7 residue are the dominant factors for determining the affinity of myoglobin

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Ligand Binding to Mb E7 Mutants

-3’ ’ I H64G H64V H64L H64F H64Q

Mutation

FIG. 4. Dependence of the association equilibrium constant (K) on the size and polarity of the E7 (position 64) amino acid in sperm whale myoglobin. Logarithms of the ratio of K for a given mutant to that for native HiP myoglobin were calculated for 02, CO, methyl (M), ethyl (E), n-propyl (nP), and n-butyl (nB) isocyanide binding to five selected mutants with substitutions at position 64. The legend in the figure going from top (02) to bottom (nB) refers to the bars going from left (solid bar) to right (open bar) for each protein.

for these ligands (Fig. 4). The order of methyl isocyanide association equilibrium constants is Gly64 = Let? > ArgG4 > Phe64 = Mete4 > Va1’j4 > GlnG4 > His64, and somewhat similar patterns were observed for the other isocyanides (Tables I and II). The low values of K for Va164 myoglobin compared to those for the Leu’j4 protein demonstrate again the inhibitory effect of substitution at the P-carbon of the E7 side chain (Fig. 4). For the smaller alkyl isocyanides, the affinity con- stants for Leu’j4 myoglobin approach those of GUYED, whereas for the larger ligands (i.e. n-propyl and n-butyl isocyanide), the overall size of the Leu’j4 residue does appear to inhibit binding significantly (Table I and Fig. 4). Finally, even though the Phe side chain is -30% larger than that of Gln or His, the Phe64 protein consistently exhibits a higher affinity for isocyanides than either Gln64 or native myoglobin (Fig. 3B). This result suggests that the polarity of Gln and His also causes inhibition of alkyl isocyanide binding.

The dependence of K on the size and stereochemistry of the alkyl isocyanide ligand is shown in Fig. 3B. The observed patterns provide a rough indication of the size and steric constraints in the distal pockets of these proteins (Mims et al., 1983). For unhindered systems such as leghemoglobin, chelated protoheme in soap micelles, and Gly’j4 myoglobin, an almost linear increase in log K versus ligand size is observed due to favorable hydrophobic effects (Olson et al., 1983; Mims et al., 1983). In contrast, native myoglobin exhibits an increase in K in going from methyl to ethyl isocyanide, and then K decreases with increasing chain length and a-substitution. This latter pattern is observed for all of the mutant myoglo- bins except H64G, regardless of the size of the E7 residue (Fig. 3B and Tables I and II). Thus, the distal histidine and other large amino acids at position 64 appear to hinder the binding of all alkyl isocyanides to the same extent, presumably interacting with the first 3 atoms of the ligand in a manner similar to that observed in the structure of the ethyl isocyan- ide complex of native sperm whale myoglobin (Johnson et al., 1989). The exact dependence of the equilibrium constant on ligand size and shape appears to be governed by interactions with other residues in the distal pocket (i.e. Phe-CDl, Val- Ell, or Leu-BlO).

Steric Kinetic Barriers-The results in Table I, Fig. 3A, and Fig. 5 suggest strongly that the height of the kinetic barrier to alkyl isocyanide binding is governed primarily by the size of the amino acid at the E7 position. The order of the association rate constants for the smaller alkyl isocyanides is Gly64 > Va164 = Leu64 > Met64 > Phe64 = His64 which is roughly the order of the side chain volumes (Fig. 5). The association rate constants for the H64R and H64D mutants are also quite large, and these results further support the idea that charged residues at the E7 position point out toward the solvent creating a more open channel to the heme iron atom than is present in the native protein.

The extremely large association rate constants for the H64G mutant provide strong independent support for the role of His-E7 as a gate to entry into the distal pocket. Johnson et al. (1989) have shown that the imidazole side chain swings out toward the solvent and away from the iron atom when ethyl isocyanide is bound to native sperm whale myoglobin. They proposed that this motion may limit the observed bi- molecular rates of ligand binding. Gibson et al. (1986) had previously shown that the rate-limiting step for isocyanide binding is migration from the solvent into a region near the iron atom and not the speed of bond formation. The obser- vation that Gly64 myoglobin exhibits kinetic properties similar to those of unhindered model hemes supports these previous conclusions. The suggestion of Johnson et al. (1989) of a steric role of Hisa in inhibiting the rate of alkyl isocyanide binding is further supported by the observation that insertion of Phe at the E’7 position results in decreased isocyanide association rate constants which are similar to or less than those of native myoglobin whereas the insertion of Val results in rate con- stants with intermediate values (Table I, Fig. 3A, and Fig. 5).

Polarity and Water Bound to HS4-One of the more strik- ing results in Tables I and II and Fig. 5 is the small dependence of the O2 association rate constants on the size of the E7 amino acid. All of the mutants except H64Q exhibit ko, values 5-20 times greater than that of native myoglobin. Even though the size of Gln is less than or equal to that of Met, Leu, and Phe, the value of &, for the Gln64 protein is only 70% greater than that for native myoglobin. These results suggest strongly that the hydrogen bonding potential of the side chain at position 64 contributes significantly to the overall kinetic barrier for O2 binding.

n 02

El co

q M

QE

cl nP

0 nB

-I H64G H64V H64L H64F H64Q

Mutation

FIG. 5. Dependence of the association rate constant (12’) on the size and polarity of the E7 (position 64) amino acid in sperm whale myoglobin. The legend and bar symbols are the same as those in Fig. 4. In this case, each bar represents the logarithm of the ratio of k’ for the mutant to that of native His” myoglobin.

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Ligand Binding to Mb E7 Mutants 3175

The CO association rate constants exhibited a similar de- pendence on substitution at the E7 position (Fig. 5). Only in the case of the H64Q mutant does the &o value approach that of native myoglobin; for all of the other E7 mutants, lo- to 50-fold increases were observed. These roughly parallel effects on the rates of O2 and CO binding were initially surprising since Gibson et al. (1986) have suggested that 02 binding is limited by the rate of ligand migration into the distal pocket whereas the rate-limiting step for CO binding is iron-ligand bond formation from within the distal pocket. Thus, a decrease in the kinetic barrier to ligand entry into the distal pocket should cause large increases in rlz& but have little effect on k&o which is given by the final rate of bond formation times the equilibrium constant for CO migrating to a location near the heme iron atom (Ansari et al., 1986). The large increases in both k&, and k&, when Gly or apolar residues are substituted for His64 argues that the polar nature of the distal histidine causes inhibition of both the rate and equilibrium constant for ligand movement into the distal pocket. This idea is supported by the results of Braunstein et al. (1988) who have shown that Glye4 and native myoglobin exhibit the same rates of geminate bond reformation at 300 K and that the increase in k& for the mutant appears to be due to an increase in the equilibrium pocket occupancy factor.

The simplest mechanistic interpretation of this polarity effect on the association rate constants comes from the crys- tallographic structures reported by Phillips (1980, 1981) and Kuriyan et al. (1986). A water molecule is found at 100% occupancy in the distal pocket of sperm whale deoxymyoglo- bin and appears to be hydrogen-bonded to the N,-amino nitrogen of His 64 This water molecule is not present in the . liganded complexes. Displacement of this H20, either to the back of the distal pocket or out of the protein, must contribute to the overall kinetic barrier for ligand binding. The bound water should also inhibit the motion of HisG4 by increasing its effective size and may form part of a hydrogen bonding lattice which extends from the distal pocket to exterior solvent (Phillips, 1981). The relatively small CO and O2 association rate constants for the Gin@ protein can be accounted for by assuming that water is also bound (albeit more weakly) to the amide nitrogen of GlnG4 in the deoxygenated form of this protein.

This interpretation is supported by kinetic studies with site-directed mutants of human hemoglobin. A water molecule is also found in the distal pocket of cy subunits in deoxyhe- moglobin, although the occupancy level is less than that observed in deoxymyoglobin (Fermi et al., 1984; Perutz et al., 1987). The His-E7 to Gly mutation in R-state a subunits, as in myoglobin, produces -lo-fold increases in k& and k&o (Mathews et al., 1989). In the case of /3 subunits, no water is found in the distal pocket of the unliganded protein, and the His-E7 to Gly mutation is without effect on the association rate constants for CO and O2 binding to the R-state form of this subunit. This view is supported by the absolute values of the association rate constants for R-state o( and /3 subunits. The observed association rate constants for O2 and CO bind- ing to /3 subunits (k&, - 1 X 10’ M-’ s-’ and k&o - 1 X lo7 M-’ s-‘; Mathews et al., 1989) are near the diffusion-controlled limits which are defined experimentally by the rate constants for unhindered model heme compounds (Collman et al., 1983; Traylor et al., 1985b). The corresponding association rate constants for R-state 01 subunits are 2- to 3-fold smaller than those for p subunits but are still 2- to 5-fold greater than those of sperm whale myoglobin. Thus, there appears to be an inverse relationship between the occupancy levels of distal

pocket water molecules near His-E7 and the CO and 0, association rate constants.

Comparisons with Naturally Occurring Proteins Containing Gin, Val, and Leu at the E7 Position-The kinetic and equi- librium parameters for O2 and CO binding to the H64Q, H64V, and H64L mutants are compared to those for Asian elephant myoglobin (Gln-E7), Aplysia myoglobin (Val-E7), and Glycera HbII (Leu-E7) in Table III. Parkhurst et al. (1980) and Mims et al. (1983) used the equilibrium constant for O2 binding to monomeric component II of G. dibranchiata to argue that in sperm whale myoglobin His64 stabilizes bound O2 by hydrogen bonding. The O2 affinity of the Glycera protein is lo-fold lower than most mammalian myoglobins, and this decrease is due to an extremely large dissociation rate constant (-2000 s-l). Glycera HbII also exhibits abnormally large association rate constants for the gaseous ligands, particularly for CO. All of these functional characteristics were reproduced by substituting Leu for HisG4 in sperm whale myoglobin. Thus, there can be little doubt that it is the aliphatic nature of the E7 residue in Glycera HbII which causes its unusual kinetic and equilibrium properties (Table III).

In Asian elephant myoglobin, Gln is present at residue 64 (E7), but the observed rate parameters for O2 and CO binding to this naturally occurring protein are very similar to those of sperm whale myoglobin. Bartnicki et al. (1983) have inter- preted this result to mean that the amide proton of GlnG4 also forms a hydrogen bond with bound OZ. Single substitution of Gln for His? in sperm whale myoglobin does cause a signifi- cant drop in O2 affinity due primarily to a lo-fold increase in the dissociation rate constant. However, the decrease in Ko, is only 5-fold compared to the 60-fold decrease observed for the H64L mutation. Thus, some stabilization of the FeOz complex occurs in sperm whale myoglobin containing Glne4, but the magnitude of this effect is smaller than that for Asian elephant myoglobin. In the latter protein, substitutions at other residues must orient the Gln-E7 into a conformation more favorable for interactions with the bound ligand (Krish- namoorthi et al., 1984, a and b).

The effects of the H64Q mutation in sperm whale myoglo- bin are similar to those reported for the same His-E7 to Gln substitution in R-state a subunits of human hemoglobin (Mathews et al., 1989). For both proteins, k& increases 1.5- to 2.0-fold, ko, increases 5- to lo-fold, and Ko, decreases 3- to 5-fold. This result strengthens the conclusions of Olson et al. (1988) that the distal protein-ligand interactions of sperm whale myoglobin and human hemoglobin a subunits are very similar.

In contrast to the results for Glycera HbII, there is little correlation between the functional behavior of the H64V mutant and that of A. limacina myoglobin which also contains a Val at the E7 position. Aplysia myoglobin exhibits a 6-fold increase in the O2 dissociation rate constant which results in a moderate decrease in oxygen affinity when compared to native sperm whale myoglobin. These changes are in the direction expected for the loss of polarity and hydrogen bond- ing potential for the E7 side chain. However, they are small compared to those observed for the H64V mutant which exhibits a lOOO-fold increase in ko,, a lo-fold increase in k&, and a net lOO-fold drop in oxygen affinity. The CO binding parameters for Aplysia myoglobin are remarkably similar to those of native sperm whale myoglobin, whereas the H64V mutant exhibits significantly more rapid rates of association and dissociation. All of these results suggest that the distal pocket of Aplysia myoglobin is able to compensate for the presence of an apolar residue at E7 and can still stabilize

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3176 Ligand Binding to Mb E7 Mutants

bound O2 and discriminate against CO as evidenced by the relatively low ratios of Kc0 to Ko, (M-values in Table III). The structural origin of this effect is not clear from the high resolution structure of the ferric form of Aplysia myoglobin reported by Bolognesi et al. (1989). These authors have spec- ulated that Arg-El0 may help stabilize polar iron-ligand com- plexes, but the side chain of this residue is undefined in the electron density maps indicating multiple conformations or mobility.

Conclusion+-Detailed mechanistic interpretations of the kinetic results in Tables I-III cannot be formulated quanti- tatively until individual rate parameters for ligand migration into and out of the distal pocket and bond formation with the iron atom are determined by nanosecond and picosecond laser photolysis techniques (i.e. Rohlfs et al., 1988; Jongeward et al., 1988). Similarly, the x-ray structures of the unliganded and liganded mutants need to be determined before the equi- librium constants can be used to assign free energy contribu- tions to specific protein-ligand interactions. All of these stud- ies are in progress. However, a number of important general conclusions can be made from the overall rate constants and must be included in any mechanism of ligand binding to myoglobin. First, the rates and extent of isocyanide binding to sperm whale myoglobin are governed by the size and stereochemistry of the E7 side chain. Second, the association rate constants for CO and O2 binding to native myoglobin are governed primarily by the polar nature of His’j4. This effect is probably due to the presence of a water molecule which is tightly bound to HiP in the distal pocket of the deoxygenated protein and which must be displaced when ligands are bound. Third, the equilibrium constant for O2 binding is determined primarily by the ability of the E7 residue to stabilize the polar iron-oxygen complex by hydrogen bonding whereas the equi- librium constants for CO and alkyl isocyanide binding are influenced by both steric hindrance and polarity effects. Fourth, regardless of the exact mechanistic interpretation, it is clear that HisE4 comprises a major portion of the gate or channel which limits the rate of ligand movement into the distal pocket of sperm whale myoglobin.

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SligarR J Rohlfs, A J Mathews, T E Carver, J S Olson, B A Springer, K D Egeberg and S G

ligand binding to sperm whale myoglobin.The effects of amino acid substitution at position E7 (residue 64) on the kinetics of

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