The Kinetics of the Reactions of Parasponia

THEJOURNAL OF BIOLOGICAL CHEMIsTllY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261,No. 29, Issue of October 15,pp. 13624-13631,1986 Printed in U.S.A.

The Kinetics of the Reactions of Parasponia andersonii Hemoglobin with Oxygen, Carbon Monoxide, and NitricOxide* (Received for publication, October 15,1985)

Jonathan B. Wittenberg$ and BeatriceA. Wittenberg4 From the Department of Physiology and Biophysics,Albert Einstein College of Medicine, Bronx, New York 10461

Quentin H. Gibson9 From the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

Michael J. Trinick and CyrilA. Appleby From the Division of Plant Industry, Commonwealth Scientificand Industrial Research Organization, Canberra,Australian Capital Territory 2601,Australia

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain C P 283. Therate of oxygen dissociation fromParasponia hemoglobin increases about 12fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting valueof 14.8 s-’. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 x 10’ M” s” at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH.The oxygen affinity of Parasponia hemoglobin, Pbo= 0.049 torr at 20 OC, calculated from the At alkaline pH kinetic constantsat pH 7, is very great. there is a prominent geminate reaction with oxygen and nitricoxide, with both subnanosecond and tensof nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantumyield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a hemelinked group withpK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 Supported by Research Grants PCM 83-03698 and PCM 8416016 from the National Science Foundation. Research Career Program Awardee 1-K6-733 of the United States Public Health Service, National Heart, Lung and Blood Institute. To whom reprint requests should be addressed Dept. of Physiology and Biophysics, Albert Einstein College of Medicine, New York, NY 10461. §Supported by Grant HL19299 from the United States Public Health Service. II Supported by Research Grants GM 14276from the United States Public Health Service and DMB 83-08454 from the National Science Foundation.

Plant hemoglobins (1) are found in nitrogen-fixing nodules formed by symbiotic associations between Rhizobium (a bacterium) and legumes ( l ) , between Rhizobium and the nonlegume Parasponia (2, 3), and between Frankia (an actinomycete) anddiverse woody dicots (4-6). Legume hemoglobins have been studied extensively; the structures of soybean (7) and lupin (8) leghemoglobin have been determined. In each a histidine residue is the proximal ligand to the heme iron atom, and anotherhistidine residue, located distal to theheme plane, enjoys unusual mobility (9-13) and may form a hydrogen bond to the bound oxygen molecule of oxyleghemoglobin (13, 14) or may ligate the iron atom of ferric leghemoglobin (9). The very great oxygen affinity of soybean leghemoglobin is achieved by extraordinarily rapid combination with oxygen together with a moderate rate of oxygen dissociation (13, 15, 16). Parasponia, a medium size (up to 20 m) tropical tree native to theMalay Archipelago region and belonging to theUlmaceae or elm family, forms the only known nitrogen-fixing, symbiotic association between a non-legume and the bacterium Rhizobium (2, 17). Parasponiu is nodulated by strains of Rhizobium that also nodulate certain members of the Leguminoseae (17). The hemoglobin found in Parasponia nodules is a plant gene product (3, 18).Parasponia andersonii hemoglobin I is a dimer of two identical polypeptide chains of 161 amino acids, corresponding to a monomer molecular weight of 18,684 (including the heme group) (3, 19).’ The amino acid sequence’shows extensive homology with lupin and soybean leghemoglobins (19). The secondary structure predicted from the amino acid sequence is similar to those of lupin and soybean leghemoglobins (19). Parasponia hemoglobin has 3 histidine residues, in contrast to thesoybean leghemoglobins which have2 and lupin leghemoglobin I1 which has 5. His-104 is considered to be the proximal ligand to the heme iron atom; His-69 may lie distal to the heme plane; an additional histidine, His-113, analogous to histidine G3 in lupin leghemoglobin (8), is a candidate for one of the titratablegroups to be described. We report that Parasponia hemoglobin shares the high oxygen affinity of soybean leghemoglobin, in each instance achieved by very rapid combination with oxygen together with a moderate rate of dissociation. Parasponia hemoglobin has heme-linked protonatable groups controlling, respectively, the A recent determination of the gene sequence of Parmponiu Hb I (Landsmann, J., Dennis, E. S., Higgins, T. J. V., Appleby, C. A., Kortt, A. A., Peacock, W. J. (1986) Nature, in press) corrects some minor errors in the amino acid sequence determined by Kortt et al. (19). The corrected chain length and molecular weight are used here.

13624

Reactions of Parasponia Hemoglobin

13625

The pH dependence of oxygen dissociation was determined using rates of ligand combination (pK 5.4-5.8) and therates of oxygen a pH-jump procedure described previously (13). As a test of the dissociation and geminate recombination (pK 6.4). Only a validity of this procedure, the rate of oxygen replacement by CO at single heme-linked titratable group, with pK 5.5, controlIing constant pH, pH 5.0, was found the same as that determined by the oxygen dissociation, is found in soybean Lb2 a (13). pH-jump procedure. EXPERIMENTALPROCEDURES

Parasponia Hemoglobin-The major hemoglobin, hemoglobin I, the only component studied inthis work, was isolated from fresh nodules of P. andersonii Planch infected with Rhizobium strain CP283 and purified to homogeneity as described (3). Stock solutions of HbOz (2 mM) in a buffer containing 100 mM potassium chloride, 1mM EDTA, and 2 mM potassium phosphate buffer, pH 7.4, were stored frozen under liquid nitrogen. After thawing, the hemoglobin remained entirely oxygenated; no ferric protein was detected in optical spectra. Buffer-A succinic acid/potassium phosphate system (13) was chosen because it buffers adequately over the desired pH range, neither constituent appears to react with Parasponia Hb, and all constituents are available as pure, fully deuterated substancesfor use in parallel experiments using NMR. The mixture contained 100 mM succinic acid (Fluka, purissima grade), 100 mM potassium dihydrogen phosphate (J.T. Baker Chemical Co., ultrex grade), and 1mM EDTA (Fluka, purissima grade) adjusted to pH7.40 or otherdesired pH with sodium hydroxide (J. T. Baker Chemical Co., ultrex grade). Molecular Size of Parasponia Hemglobin-Apparent molecular size was determined by chromatography at 4 "C on acalibrated column of Sephacryl S-200,1.6 by 60 cm, developedwith air-saturated 0.1 M potassium phosphate buffer containing 1 mM EDTA, pH 7.2, for HbOz and ferric Hb or developed with argon-equilibrated buffer containing 1 mM sodium dithionitefor ferrous Hb. The sample volume was 2% of the column volume. From 500 p~ samples, as loaded, peak effluent concentration was about 200pM; from 5 flM samples, as loaded, peak effluent concentrationwas about 1pM. Good separation in the range of molecular weights from 13,000 to 150,000 was achieved; Kav for the calibrating proteins ribonuclease (13,700), chymotrypsinogen (25,000), ovalbumin (43,000), bovine serum albumin (67,000), aldolase (158,000) were 0.55, 0.46, 0.32, 0.24, and 0.14, respectively. pH-dependent Change in Optical Spectra-Optical titratious were et al. (13) in a Hitachi performed a t 20 "C as described by Appleby -~ Perkin-Elmer model 557 spectrophotometer interfaced to a PDP 11/ 03 computer and Hewlett-Packard model 7221-B plotter. Ligand Reaction Rates Using Stopped-Flow-A Gibson-Milnes (20) stopped-flow apparatus with a 2-cm light path in the observation cell and equipped with a log ratio amplifier was used to measure ligand dissociation rates and the rates of carbon monoxide combination. Data were accumulated in a Biomation model 802 transient recorder and subsequently plotted using an X-Y recorder. The change in optical density ofveryslow reactions was plotted directly on a Hewlett-Packard model 7004B X-Y recorder. Oxygen Dissociation Rate-Solutions of Parasponia HbOp (3 p M HbOz, 2.8 PM free oxygen in buffer) were mixedrapidly with solutions of carbon monoxide (approximately 1mM) in buffer and thereaction followed a t 417 nm, the absorption maximum in the difference spectrum Parasponia HbCO minus HbOp. The reaction was homogeneous and followed first order kinetics to more than 90% completion. The rate was independent of carbon monoxide concentration (>0.5 mM carbon monoxide). The rate measured a t 403 nm, the minimum in the difference spectrum, Parasponiu HbCO minus HbO2, was the same as that measured a t 417 nm. In a few experiments, dithionite (5 mM in buffer before mixing) was used to trap oxygen liberated from Puraspnia HbOz and the reaction was followed at 431 nm, a maximum in the difference spectrum: Parasponia deoxy-Hb minus HbO,. Although this reaction followed a somewhat inhomogeneous course, the initial rates were the same as those measured using carbon monoxide. In one experiment, a solution of Parasponiu HbOo (31 p~ HbOz, no added free oxygen) was mixed rapidly with a solution of carbon monoxide (1 mM in buffer) and the reaction followed a t 577 and 560 nm, a maximum and minimum, respectively, in the difference spectrum: Parosponia HbOZ minus deoxy-Hb. ~

The abbreviations used are: Lb, legume hemoglobin or leghemoglobin; Hb, the hemoglobin component I (the only component used in thiswork) isolated from Parasponia andersonii root nodules; HbO,, oxy Parasponia hemoglobin; HbCO, carbon monoxy Parasponia hemoglobin.

CarbonMonoxideDissociation Rate-Solutions of Paraspnia HbCO (prepared by adding 2 mM HbOp to a solution containing 8 p M CO and 17 p~ dithionite in buffer to give a mixture containing 3pM HbCO, 5 p~ free CO, and 5 p~ residual dithionite) weremixed rapidly with oxygen-free solutions of nitric oxide (approximately 2 mM) in buffer. The reaction was followed at 417 nm, an absorption maximum of Parasponia HbCO. The reaction was homogeneous and followed first order kinetics to about 95% completion. CarbonMonoxideCombinationRate-Solutions of Parasponia deoxy-Hb (0.5 p~ Hb, 125p M dithionite in buffer) were mixedrapidly with solutions of carbon monoxide (2.5 to 20 p M in buffer containing 125 GM dithionite), and thereaction followed at 417 nm. The reaction course was first order and homogeneous to more than 90% completion. The rate measured at 430 nm, a minimum in the difference spectrum Parasponia HbCO minus deoxy-Hh, was the same 9s that measured at 417 nm. Ligand Recombination Rates Following Flash Photolysis-Ligand recombination following flash photolysis was measured spectrophotometrically as described by Sawicki and Gibson (21, 22). Most measurements were made a t 20 "C at a heme concentration near 70 p~ in 1-mm cells, using a 300-11s flash. Data were collected with several levels of photolysis light centered a t 575 nm, the highest level being just below the point at which artifactual changes set in. The effective rate of work, based on a quantum yield of 1, was about 5 X los s-'. Data collection used a Biomation 805 transient recorder coupled to a PDP 8F computer. The observing light was provided by a 75-watt xenon arc, screened with a blue glass filter before reaching the sample. To measure CO binding, about 1 ml of buffer was placed in a 100ml tonometer with a 1-mm cell fused to the bottom. The tonometer was flushed for some minutes with purified nitrogen and then with CO. A suitable volume of Parasponia HbOz was then introduced, and the fluid equilibrated with the gas phase. Most measurements were made at 432 nm, a wavelength near the absorbance maximum of deoxy-Hb. An excellent isosbestic was found at 422 nm. The time course of the absorbance excursion was accurately first order and there was no slow absorbance change at 419 nm, where the difference between HbO, and HbCO is large. This indicates that there was no interference by residual oxygen. A comparison between the combination of carbon monoxide with Parasponia Hb as measured by stopped-flow and by flash photolysis on the same solution was carried out using the flow-flash apparatus described by Ludwig and Gibson (23). Deoxy-Hb and carbon monoxide were first mixed in the stopped-flow mode and the reaction followed using a Biomation 805 recorder. Then, carbon monoxide was dissociated from the same portion of solution which remained a t rest in the observation tube by a laser flash using Rhodamine 575. The laser light was excluded from the photomultiplier by two thicknesses of Corning blue glass 7-57. The recombination of CO with the hemoglobin was then followed as before. The second order rate constants calculated for the two procedures agreed within 2%. Measurements of oxygen binding were made at 436 nm, using airequilibrated solutions. The kinetic difference at pH5.1 and 7.0, using the most intense photolysis light, was 89% of the static difference HbO, minus deoxy-Hb (dithionite added). An excellent isosbestic was found at 417 nm, suggesting that only two absorbing species were present throughoutthe experiment. An example of one kinetic run at pH 9.01 is presented as Fig. 1 which shows three data sets collected with maximal light and one set with the light attenuated 32-fold. The data are nearly first order, although theoretically the points should lie on a gentle curve as the oxygen concentration of the solution changes. The difference in slope of the two lines is largely accounted for by the larger amount of oxygen released into solution by the brighter flash, with second order rates of 2.04 and 1.90 x lo8 M" s" for the examples shown. Geminate Oxygen Recombination-The geminate reactions were followed by flash photolysis using a 25-ns pulse centered at 575 nm, with an energy of 50 mJ. In theoptical system used, this was sufficient to cause dissociation of oxymyoglobin at a rateof 5 ns". The reaction was followed using a 75-watt Xe arc lamp, pulsed for 0.1 ms to 20 times normal brightness. The light passed through a Spex monochromator and was monitored using a 1P28 photomultplier working into 50 ohms. The signals were measured with a Hewlett-Packard


13626

0.5 0.3

0.1

d U 0.05

0.01 i I / T ) r103

TIME ( p s e c )

( I / T ) x IO3

FIG.1. Oxygen combination following flash photolysis of Parasponia Won.95 PM heme, pH 9.01, 20 "C,1-mm path.Solid circles, open circles,solid squares, maximal photolysing light; solid triungles, light attenuated 32-fold. Different symbols represent different experiments. The second order rate constants are 2.04 and 1.90 x 10s M" S-'. FIG.2. Effect of temperature on the rates of combination of Parasponia deoxy-Hb with oxygen (solid circles) and with carbon monoxide (open circles). The heats of activation found with oxygen and carbon monoxide are, respectively, 3.7 and 3.7 kcal/mol. FIG.3. Effect of temperature on the dissociationof oxygen (solid circles) and carbon monoxide(open circles) from Parasponia W.The heatsof activation found with oxygen and carbon monoxide are, respectively, 23 and 18 kcal/mol.

TABLE I

combination, CO combination, 0, dissociation, and CO dissociation, respectively. pH Dependence of Reaction Rates-The pH dependence of The subunit molecular weight is 18,6841. the oxygen combination rate constant between pH 4 and 7 is Apparent molecular weight Initial best represented by assuming a single ionizing group of pK hemoglobin 5.37, with theoretical limiting values of 0.133 and 1.65 X lo8 Ferric Hb HbO2 concentration Deoxy-Hb M" s" for the acid and alkaline forms, respectively (Fig. 4). PM There is an unexplained discontinuity with an increase in 5 34,000 34,000 36,000 rate at pH values greater than pH 7. 500 36,000 33,000 34,000 The pHdependence of the oxygen dissociation rate fits the relation for a single ionization of pK 6.41 with limiting values oscilloscope model 1725 (275 MHz). The photographs were traced, of 1.2 and 14.8 s" for the acid and alkaline forms respectively digitized, and converted to absorbance changes. Most observations (Fig. 4). were made at 432 nm. The pH dependence of carbon monoxide combination fits the relation for a single ionizing group of pK5.75 with limiting RESULTS values of 2.0 and 13.7 X lo6 M" s" for the acid and alkaline Parasponia Hemoglobin Is Dimeric-Appleby et al. (3), on forms (Fig. 5). Carbon monoxide dissociation is independent the basis of limited experiments, suggested that Parasponia of pH from pH 5.0 to 8.5. Hb I was probably a dimer. Chromatography of dilute (iniNitric oxide combines somewhat more rapidly than oxygen, ) concentrated (initially 500 p ~ Parasponia ) tially 5 p ~ and kl = 240 X lo6 M" s" at pH9 (Table 11). Change to acid pH Hb ona Sephacryl S-200 column establishes that Parasponiu produced a marked drop in the effective quantum yield; referric Hb, deoxy-Hb, and HbOz exist as concentration-indecombination could not be measured accurately. pendent dimers (Table I). The apparent pK values of these heme-linked ionizations Rates of Ligand Combination and Dissociation-Rates of are reported in Table 111. these reactions at the alkaline limits (pH 7-9) of the pHpH-dependent Change of Optical Spectra-A pH-dependent dependent change of their principal titratable component are change observed in the visible region of the optical spectrum presented in Table 11, where they are compared to the rates of reaction of soybean leghemoglobin a. The rate constantfor of Parasponia HbO, resembles that described for soybean oxygen binding does not change from 5 to 250 M M Hb. The LbO, (13, 24). It fits the relation for a single ionization, with rate of oxygen dissociation does not change from 1 to 31 pM pK 6.23 (Fig. 6). A smaller, pH-dependent change involving the absorbance maximum of Parasponia deoxy-Hb in the HbO,. Temperature Dependence of Reaction Rates-The temper- visible region wasalso observed. It fits the relation for a single ature dependence of ligand combination is small (Fig. 2); that ionization with pK 5.65 (Fig. 6). Geminate Combination of Ligands to Parasponia Hemogloof ligand dissociation is about 5-fold greater (Fig. 3). The heats of activation are 3.7, 3.7, 23, and 18 kcal/mol for 0, bin-The results with Parasponia hemoglobin are unusual, Apparent molecular weightof Parasponia hemoglobin determinedon a column of Sephncvl S-200

13627

Reactions of Parasponia Hemoglobin TABLE I1 Kinetic and equilibrium constants for the reactions of Parasponia Hb with ligands, comparedto those of soybean Lb a Values are taken at the alkaline limit of pH-dependent change: pH 9 for Parasponia Hb except for oxygen combination, where the value at pH 7 is taken, and pH 7.0 for soybean Lb. Equilibrium constants are estimated from the kinetic constants. All data were obtained at 20 "C. Data for soybean Lb a are from Appleby et al. (13). Carbon

Oxygen

k 'om s-1 165 X 10' 116 X 10' "1

s-1

monoxide

K' (=k:k')

Pm

1'on

ItM

torr

M" s-l 13.7 X 10' 12.7 X 10'

&tJ S-1

L ' (=l:l')

PM

nM

torr 1.0X 10-3 0.45 X

Partition coefficient" M = [HbCO] pOJ w b 0 2 1 PC0

Nitric oxide

k', "1

s-l

50 240 X 10' 1.4 0.0187 14.80.049 89 Parasponia Hb 0.0078 0.62 58 Sovbean Lb 5.55 0.026 48 These numbers are calculated in terms of the gaseous pressures; alternatively, one could define M' in terms of the ratio of the two equilibrium constants LIK, in which case M' = 1.34 M at 20 "C. ~~~

TABLE I11 Apparent pK of hem-linked proton-dependent change in Parasponia Hb compared to those of soybean leghemglobin a The pHrange examined is pH 4.6 to 9.0. Apparent pK Pamsponio Hb

pH

FIG. 4. Kinetic constants for the reactions of oxygen with Parasponia deoxy-Hb as functions ofpH at 20 "C. Solid circles, second order rate constant,k', combination; open circles, k, dissociation. The lines are computer-assisted best fits to the relation for single ionizations. The midpoints are: pK = 5.4 for combination and pK = 6.4 for dissociation.

Soybean Lb

Deoxyhemoglobin Optical spectrum 5.65 Invariant&& O2combination 5.37 Invariantb# CO combination 5.75 Invariantb NMR Invariant' Oxyhemoglobin 6.23 5.43'b Optical spectrum Oz dissociation 6.41 5.46b8d Geminate O2recombination 6.5 NMR 5.5' Carbon monoxide hemoglobin Optical spectrum 4.3" Invariant' CO dissociation Invariant Invariantb Fuchsman and Appleby (24). Appleby et al. (13). Complex changes are observed above pH 8 and below pH 5. Ikeda-Saito et al. (14). e NMR is of meso heme proton resonances (13) and of the distal histidine (25, 26). 'Almost invariant above pH 5. Change occurs at more acid pH.

pH

FIG. 5. Second order rate constant, 1'. for the combination of carbon monoxidewith Parasponia Hb as a function ofpH at 20 "C. pK = 5.75. Carbon monoxide dissociation is independent of pH.

though there arefew data for other hemoproteins with which to compare them. Data have been obtained for CO, 02,and NO as ligands at several temperatures and pH values. With CO as ligand, geminate combination is minimal under all the conditions examined. The largest reaction is seen at neutral or alkaline pH and low temperature. In one series of experiments the rapid reaction seen accounted for between 5 and 7% of the absorbance excursion and had a half-time of 40 ns at pH 7 and 2 "C, corresponding to a relaxation at a rate of

FIG. 6. Change in optical spectra of Parasponia HbO1 and deoxy-Hb as functions of pH at 20 "C. Solid circles, deoxy-Hb (A662nm-As12am);opencircles,HbOz (A576m-Amnm).The midpoints are pK= 5.65 and pK= 6.23 for deoxy-Hb and Hb02,respectively.

1.6 X IO7 s-'. No geminate reaction was observed at pH 7, 20 "C, nor at pH4 at any temperature. With oxygen as ligand, a well-marked geminate reaction is observed, accounting for 25% of the absorbance excursion at 20 "C and pH 7, with a half-time of 15 ns. On coolingto 2 "C, the proportion increased to 41%, buttherate was little changed. Change in pH, however, produced a marked effect


13628

with progressive disappearance of geminate combination as the solutions were made more acid. A sample of oscilloscope records illustrating this effect is shown in Fig. 7. In addition, the effective quantum yield at the end of the flash is only about half as great at pH 4 as at pH 9. This is not due to denaturation of the protein in the acid solutions as a prolonged (300ns) flash gave the same absorbance change at 432 nm at either pH (lowerpanels of Fig. 7). The drop in effective quantum yield is also expressed in the obvious difference in the slopes of the reaction records. There is a good correlation between amplitude of geminate reaction and apparent quantum yield expressed in the curves of Fig. 8. The two upper curves show the absorbance excursion at the end of the photolysis pulse expressed in arbitrary unitsfor two different levels of light intensity. The lowest curve is the amplitude of the geminate reaction, measured as the difference between the peak height and the level reached by the trace after 200 ns, as in the upper right panelof Fig. 7. All three curves have half-effect at pH6.5. DISCUSSION

"

5.0

7.0

6.0

9.0

pH

FIG. 8. Effect of pH on geminate combination of oxygen with Parasponia Hb.A solution of Parasponia HbO, equilibrated with room air, 75 p~ in heme in a 1-mm cell, wasphotolyzed at 20 "C with a 25-ns pulse. The reaction was followed at 432 nm at each of the pHvalues shown. The uppermost curve shows the excursion when the middle curve to %.The the laser pulse was attenuated to %, meausrement of the excursion was made 200 ns after the flash. The maximum excursion shown corresponds to theremoval of 45% of the total oxygen. The lowest line is the difference between the peak in deoxyhemoglobinat the end of the flash and thelevel 200ns later for the 1h attenuation level and is a measure of geminate recombination. The hemoglobin concentration varied slightly between pH values and the recorded values have been adjusted for this; thus the arbitrary scale of the ordinate has thesame relative value for all lines.

Parasponia Hb is a dimer formed of two identical subunits each bearing one heme. The dimer does not dissociate even in very dilute solution (Table I). The rate constantsfor oxygen combination and dissociation are each independent of protein concentration, and the progress of the reactions with excess oxygen and carbon monoxide are closely first order throughout most of their course. This indicates that each heme group The reactions of Parasponia Hb with carbon monoxide are reacts independently without intra- or intermolecular heme10 to 1000 times slower but in general parallel those with heme interaction. The kinetics of the overall reactions of Parasponia and oxygen. The rates of reaction with carbon monoxide are very soybean hemoglobins with oxygen are strikingly similar (Ta- similar to those of soybean Lb (Table 11). This is reflected in ble 11). Oxygen combination is very rapid and, at thealkaline the partitions of Parasponia and soybean Hb between oxygen limit, approaches within a decade the expected diffusion- and carbon monoxide ( M of Table 11), which are nearly the limited rate. The rates of oxygen dissociation are moderate. same. Although it has long been recognized that thecombination The resulting oxygen affinities, Pb0is 0.049 and 0.026 torr for Parasponia and soybean hemoglobin, respectively, are ex- and dissociation of ligands with hemoproteins must be a complex process, ithas only recently become possible to traordinarily great. demonstrate and measure some of the substeps involved in these reactions (27-33). The reactions are of interest in themselves, but, as the structures of several leghemoglobins have 0.1 been determined with high resolution, it seems particularly desirable to examine Parasponia hemoglobin in this respect. The reactions with oxygen have been analyzed further by assuming the minimal scheme,

-

' u)

f: 0.0

k, k3 k, A-k,B'k,C.k,D

0.3

0.0

Time FIG. 7. Geminate combination of oxygen with Parasponia

Hb.The records were obtained with a 25-11s pulse (top panels) and with a 300-11s pulse (lower panels) with energies of 50 and 300 mJ, respectively. The time scale was 50 ns/division throughout. The multiple traces in each panel show the results obtained when the laser flash was either unscreened or attenuated by a factor of 2,4, or 8 by passage through a neutraldensity filter. In each panel the largest trace was obtained with the unscreened flash. The absorbance at 432 nm is always greater immediately after the flash. In the two lower panels the top of the grid corresponds to zero light. A back-off voltage was employed for the two upper panels. The solutions contained 75 p~ heme, were equilibrated with air, and were examined in a 1-mm cell at 20 "C.

where A represents liganded Parasponia hemoglobin, and D is free deoxy protein in equilibrium with ligand in solution. The species B and C are intermediates in which a ligand molecule remains associated with the protein butis not bound to theheme. The rate k, is the rate of breaking the Fe-ligand bond in the light; a quantum yield of unity is assumed. The first step in the reaction with ligand free in solution, D to C , is not taken into account as it is too slow, even with Parasponia hemoglobin, to make a significant contribution to the absorbance changes observed. The remaining rates have been assigned by fitting the family of absorbance changes obtained with several flash intensities to the integrated form of the rateequations derived from the scheme, as described by Gibson et al. (33). The four undefined parameters can often be determined satisfactorily by such data, depending on the actual values in a given case. In general, the reaction B to A, defined by kz,is rapid, and the population of B is roughly kl x A/(k, k.J, giving a starting value for the curve-fitting

+

Reactions of Parasponia Hemoglobin procedure. The geminate reaction consists of two parts, avery rapid one as the flash shuts off and A is repopulated from €3 with rateconstant k f anda slower reactionas species c repopulates A through B. The approximate rate constant for this reaction is k4 k,. The amplitudes of the two components are determined by k, which largely governs the population of C during the photoflash with small values giving a large rapid reaction, and larger values a greater proportion of the slower one. Values of the parameters derived from curve fitting are given in Table IV. They show, first, that therate of rebinding of ligand, expressed by kz, though always large, is also very variable, acharactershared with k4. Allof thedataare satisfactorily represented with quite similar values of 123 and k,, suggesting that these parametersdescribe a diffusion process as proposed by Marden (27). Two examples of the results of curve fitting areshown in Fig. 9, A and B . When thereaction with nitric oxide wasstudied in the same way, analogous results were obtained, the chief difference at alkaline pH being that a much larger value of k2 was required than with oxygen. At acid pH so little ligand was dissociated at any stage of the reaction, corresponding at the peak to a change of 2-3% in saturation, that the assignment of rates

+

TABLEIV Rate constants for ligand binding to Parasponia hemoglobin The rate constantsapply to the A $ B C + D scheme given in the text. The values in parentheses were derived as described in the text. The remaining values were obtained by nonlinear least squares minimization comparing the solutions to the rateequations with the observed results. The root mean square residuals lay between 0.02 and 0.04 in saturation. The uncertainties in theparameters calculated from the covariance matrix were unrealistically small as compared with the results in separate experiments, perhaps because the flash intensity was assumed to be known precisely, and have not been quoted. With the exception of k, and k5 for oxygen at pH 4.6, all parameters were well defined. Ligand

2.2

0 2 0 2

7.0 7.0

0 2

10.70

2

4.6

NO NO (0.2) (165) NO 4.6

co

pH kz "C 20 2 0.020 1.80.042 0.015 20 0.0024.60.17 7.1 2 20 7.0 0.029 6.1 0.059 12.0 2 7.0 20 2 7.0 (0.2)(0.002)

Temperature

kh

k,

0.18 0.22

0.027

0.030

0.22 0.17 0.20

0.006

0.018

0.048

0.021

k3 M-1

(0.04)

13629

could only be done by analogy with oxygen. The entries in Table IV have been enclosed in parentheses to stress this. With carbon monoxide as ligand, the problem is just the opposite. So much ligand is dissociated and so little returns to theheme that independent determination of all the parameters is impossible. Again, an estimate of kz has been given for purposes of comparison. It ismuch smaller than the corresponding values for oxygen and nitric oxide, suggesting that the difference between ligands derives chiefly from this source, i.e. from a difference in rate of reaction at theheme. The values in Table IV can be examined for consistency with the overall values given in Table I1 since the product of C eD the binding constants for each stage of the A $ B scheme should be equal to the overall constant, i.e. k' = k2k4&/(k2k4-t k2k5 + k3ks).To make the comparison, a value is required for kl, now the dark dissociation velocity constant. This is equal to the product of the observed dissociation rate and the apparent quantum yieldfor transfer of ligand to solution. An approximate value can be obtained from the data of Fig. 7 using the amplitude after geminate recombination is complete together with the estimated numbers of quanta delivered to the sample as measured by the dissociation of carbon monoxide myoglobin. It is 0.05. The rate of dissociation of oxygen from the heme is then 300 s" at 20 "C. The resulting value for the product of the parameters is 1.2 x IO7 M-' s-'. This is very close to the overall value of 1.1 X lo7M" s-l derived from Table 11. In making the calculation, it has been assumed that there is no change in activity of ligand in passing from D to C in the scheme. At pH 4.6 and 20 "C,the value of k4 is poorly defined, but the calculated value of 1.7 x lo7M-' s-' does not differ unreasonably from the value derived from Table I1 and the data on Fig. 4 which is 2.2 X lo7. This agreement, incidentally, strongly supports the assumption that thequantum yield for the step A B is 1. The temperature dependence of the parameters in Table IV is notably slight, and in some cases (k,) negative. In this specific case, the rate constant is really described two processes, recombination, and diffusion away from the immediate region of the heme. On cooling, there is little change in the recombination velocity, but it is able to operate longer on the newly dissociated ligand which diffuses away less readily at lower temperatures. The compound parameter k, appears to increase as thetemperature is reduced. The slight temperature dependence of the rates is generally consistent with the suggestion that they arise from diffusion, but the large steps in

+

-

1.o

1P

'*or--"l

FIG. 9. Example of curve fitting procedure used to estimate parameter values in the text scheme A B + C e D. The observations were made using 85 eM Parasponia HbOz equiiibrated with room air at 20 "C,in buffers of pH 7 and 4.6 for A and E , respectively. Observation was of a 1-mm path at 432 nm. The laser pulse was used directly and attenuated to%,W, and % in A , and to Yz and lh in E . The points are observed, the lines calculated giving equal weights to all points.

8

0.a

0.8 P

I %

0.6

0.6

Q)

4

TI

-= C

0

0.4

op

0.4

0, n 0.2

0.0 0

100

50

-

Time ns.

150

0.2

a

0.0

-a -

(

50

100

lime- ns.

150

13630

Reactions of Parasponiu Hemoglobin

the rates as one goes from k2 to & and kl to ks are not consistent with diffusion in a geometrically simple volume. Comparison of the values for nitricoxide and oxygen agree with the interpretation suggested for the oxygen data since the main difference is in thevalues of $, the chemical barrier step. The geminate reaction with carbon monoxide is to small to permit assured analysis, but again the results are consistent with a description in which k2 is primarily affected. One unusual feature of the geminate reactions of Parasponiu Hb with oxygen and nitric oxide is the considerable effect of pH shown in Figs. 7 and 8 and in Table IV. There have been few studies of other hemoproteins with these ligands, and no pH effect has previously been reported. There is, however, a striking parallel between the results with oxygen a t acid and alkaline pH and the differences between human hemoglobin subunits observed by Morris et al. (34).When FeCo hybrid hemoglobins react with oxygen, a Fe subunitsshow little geminate recombinations, while with B Fesubunits geminate recombination dominates the nanosecond kinetics. With Parasponiu Hb thereaction at pH7 resembles that of B subunits, while at pH 4 it resembles that of the a subunits. All of the experiments with the hybrid human hemoglobins were performed at pH 7,however, so the meaning of the parallel is not clear. The analysis of the geminate reactions summarized in Table IV shows the effect of pH to derive from changes in the reaction B to A ($) and in thereaction C to B ( k J . The first may reasonably be associated with the heme and itsimmediate environs and perhaps with protonation of His-69, but the second change, which is at least as great, affects a diffusion step in the transitionbetween heme and theprotein internal environment and,so, presumably reflects a pH-linkedchange in conformation. Three properties of Parasponiu deoxyhemoglobin change with similar pK (Table 111).These arethe rateof combination with oxygen, the rate of combination with carbon monoxide, and the electronic configuration of the heme as reported by the optical spectrum. For the moment we consider that the three apparent pK values reflect protonation of a common single group. The effect of pH clearly is being exerted at the heme rather than by opening and closing of a hypothetical barrier to diffusion which, if rate determining with oxygen, would have little effect on carbon monoxide, which combines 10 times more slowly. In fact the ratioof rates at theacid and alkaline limits (7-%fold increase) isthe same for oxygen and carbon monoxide. Three properties of Parasponiu oxyhemoglobin change with nearly the same pK. These arethe rateof oxygen dissociation, the electronic structure of the heme as reported by the optical spectrum, and the amplitude of geminate recombination of oxygen followingphotolysis by a very brief flash. The coincidence of the apparent pKvalues suggests that protonation of the same group may govern all of these properties. It seems reasonable to consider geminate recombination together with the properties of the liganded protein, because the starting pointisthe oxygenated protein, justasit is for oxygen dissociation, and very little time is available for change in conformation in thefew nanoseconds between photolysis and recombination. We propose, as a working model, that Parasponiu hemoglobin has two different protonatable groups, one affecting predominantly the properties of deoxyhemoglobin, the other the properties of oxyhemoglobin.An alternative model with a single heme-linked protonatable group with different pK in the deoxygenated and oxygenated states would also fit the facts butwe consider it less probable because the protonation

governing the properties of soybean oxyleghemoglobin is without discernable effect on the deoxy protein (13, 14, 25, 26). The two models should be distinguished readily using NMR to identify the protonatable groups; such a study is in progress. The finding of distinguishable pK values for proton effects on deoxy and oxy Parasponiu hemoglobin, respectively, implies that thetwo protonations exert theireffects on the heme in different ways.One hypothesis, without present experimental support, is that effects on deoxyhemoglobin are exerted through the heme periphery or the proximal ligand to the heme iron, and that effects on oxyhemoglobin, as in soybean leghemoglobin (13, 14, 25, 26), are consequences of protonation of the distal histidine. Acknowledgments-We thank Laura Grinvalds, Saqui Selima Huq, and Patricia Wong for their invaluable assistance.

REFERENCES 1. Appleby, C. A. (1984) Annu. Reu. Phnt Physwl. 35,433-478 2. Trinick, M. J. (1973) Nature 2 4 4 , 459-460 3. Appleby, C. A., Tjepkema, J. D., and Trinick, M. J. (1983)Science 220,951-953 4. Davenport, H. E. (1960) Nature 186,653-654 5. Tjepkema, J. D.(1983) Can. J. Bot. 6 9 , 2924-2929 6. Fleming, A. I., Wittenberg, B. A., Wittenberg, J. B., and Appleby, C. A. (1985) Proc. A w t . Biochem. Soc. 1 7 . 4 3 7. Ollis, D. L., Appleby,C. A., Colman, P.-M.,Cutten, A. E. C., Guss, J. M., Venkatappa,M. P., and Freeman, H. C. (1983) A u t . J. Chem. 36,451-468 8. Harutyunyan, E. G., Kuranova, I. P., Vainshtein, B. K., and Steigemann, W. (1980) Kristalbgrafiya 26,80-103 9. Appleby, C. A., Blumberg, W. E., Peisach, J., Wittenberg, B. A., and Wittenberg, J. B. (1976) J. Bwl. Chem. 251,6090-6096 10. Wright, P. E., and Appleby, C. A. (1977) FEBS Lett. 78,61-66 11. Johnson, R. N., Bradbury, J. H., and Appleby, C. A. (1978) J. BwL Chem. 253,2148-2154 12. Appleby, C. A., Blumberg, W. E., Bradbury, J. H., Fuchsman, W. H., Piesach, J., Wittenberg, B. A., Wittenberg, J. B., and Wright, P.E. (1982) in Interactions Between Iron and Proteins in Oxygen and Electron Transport (1980 Symposium Proceedings) (Ho, C., ed) pp. 435-441, Elsevier/North-Holland, New York 13. Appleby, C. A., Bradbury, J. H., Morris, R. J., Wittenberg, B. A., Wittenberg, J. B., andWright, P. E.(1983) J. Biol. Chem. 2 5 8 , 2254-2259 14. Ikeda-Saito, M., Hori, H., Inubushi, T., and Yonetani, T. (1981) J. Bwl. Chem. 2 6 6 , 10267-10271 15. Imamura, T., Riggs, A., and Gibson, Q. H. (1972) J. Bwl. Chem. 247,521-526 16. Wittenberg, J. B., Appleby, C. A., and Wittenberg, B. A. (1972) J. Bwl. Chem. 247,527-531 17. Trinick, M. J., and Galbraith, J. (1980) New Phytol. 8 6 , 17-26 18. Appleby, C. A., Chen, Y. P., Dudman, W. F., Higgings, T. J., Inglis, A. S., Kortt, A. A., Sutton, W. D., and Trinick, M. J. (1984) in Advances in Nitrogen Fixation Research (Veeger, C., and Newton, W. E.,eds) p. 493, Martinus Nijhoff/Dr. W. Junk Publishers, The Hague 19. Kortt, A. A., Burns, J. E., Trinick, M. J., and Appleby,C. A. (1985) FEBS Lett. 1 8 0 , 5 5 4 0 20. Gibson, Q. H., and Milnes, L. (1964) Biochem. J. 9 1 , 161-171 21. Sawicki, C. A., and Gibson, Q. H. (1976) J. Bwl. Chem. 2 5 1 , 1533-1542 22. Sawicki, C. A., and Gibson, Q. H. (1977) J. Bid. Chem. 2 5 2 , 5783-5787 23. Ludwig, B., and Gibson, Q. H. (1981) J. Biol. Chem. 256,1009210098 24. Fuchsman, W. H., and Appleby, C. A. (1979) Bwchemistry 1 8 , 1309-1321 25. Mabbut, B. C., Appleby, C. A., and Wright, P. E. (1983) Biochim. Biophys. Acta 749,281-288 26. Trewhella, J., Wright, P. E., and Appleby, C. A. (1986) A u t . J. Chem. 39,317-324 27. Marden, M. C. (1982) Eur. J. Bwchem. 128,399-404 28. Chernoff, D. A., Hochstrasser, R. M., and Steele, A. W. (1982) in Hemglobin and Oxygen Bindings (Ho, C., ed) pp.345-350,


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Elsevier Scientific Publishing Co., Inc., New York Bowne, S. F., Doster, W., Eisenstein, L., Frauenfelder, H., 29. Henry, E. R., Sommer, J. H., Hofrichter, J., and Eaton, W. A. Reinisch, L., Shyamsunder, E., and Jung, C. (1985)J. Bwl. (1983)J. Mol. Biol. 166,443-451 Chem. 260,8803-8809 30' Hofrichter* J*v Sommerf J' H'* Henry, E' and Eaton, w'A' 33. Gibson, Q . H., Ikeda-Saito, M., and Yonehni, T.(1985)J . Bhl, (1983)Proc. Natl. Acad, Sci. U.S. A. 80,2235-2239 Chem. 260,14126-14131 31. Duddell, D. A., Morris, R. J., and Richards, J. T.(1979)J. Chem. SOC.Chem. Commun. 75-76 34. Morris, R. J., Gibson, Q . H., Ikeda-Saito, M., and Yonetani, T. 32. Stetzkowski, F., Banerjee, R., Marden, M. C., Beece, D. K., (1984)J. Bwl. Chem. 269,6701-6703