The Kinetics of the Reactions of Leghemoglobin with Oxygen and Carbon Monoxide*

values of the kinetic constants for the reactions of have been determined spectrophotometry.


SUMMARY
The values of the kinetic constants for the reactions of legume root nodule hemoglobin (leghemoglobin) with oxygen and carbon monoxide have been determined by stopped flow spectrophotometry.
Leghemoglobin a and leghemoglobin c, which differ in amino acid sequence and peptide chain length, have very similar kinetic constants.
The oxygen combination rate constant is the largest measured for any hemoglobin.
The oxygen dissociation rate constant is similar to that of mammalian myoglobins. The oxygen affinity calculated by combining the kinetic constants agrees with the equilibrium value determined directly. The rate constant for combination with carbon monoxide is large, and is reflected in the very great affinity of leghemoglobin for carbon monoxide.
The kinetic constants for the reactions of leghemoglobin with oxygen are particularly well suited to favor the facilitation of oxygen diffusion by leghemoglobin in the environment of very low mean oxygen pressure existing within the cells of the root nodule.
It is suggested that leghemoglobinfacilitated oxygen diffusion serves not only to augment the influx of oxygen, but serves as well to make the oxygen pressure within the nodule or cell everywhere nearly the same.
Two major components diffcrin g ill umlecular size and other properties nlay be isolated from soylrearr root nodulr:s: leghemoglobin  (9). Two outstanding properties of the protein are its very great affinity for oxygen (3, lo), and the concentration at which it occurs in the root nodule.
The concentration in the nodule is 0.2 to 0.5 mrvr (4), commensurate with the concentration of myoglobin in red skeletal muscles; the concentration in those domains within the nodule to which the protein is restricted must be several-fold greater.
The function of leghemoglobin in the nodule remains unknown (for reviews see Evans and Russell (11) and Wittenberg (12)), although it is clear that there is an obligatory relation between the occurrence of leghemoglobin and the ability of a nodule to fix nitrogen (13).
One question t,o which we address ourselves here is whether leghemoglobin may facilitate oxygen diffusion within the nodule.
Leghemoglobin in situ in the nodule is partially oxygenated. The balance is deoxygenated leghemoglobin exhibiting a typical myoglobin-like spectrum characterized by a broad absorption maximum centered near 555 nm (14). Leghemoglobin in situ undergoes reversible oxygenation (14). Leghemoglobin isolated from nodules fragmented into neutral or slightly alkaline media, is in the oxygenated state (6, 14). However leghemoglobin extracted at acid pH may be isolated in a ferrihemochromogen form which may be reduced (e.g. by dithionite) to a typical ferrohemochromogen (8, 15). Appleby (8) has shown that this hemochromogen is formed by reversible combination of leghemoglobin with a small ligand (called X)1 and that formation of leghemoglobin-X complexes is favored at slightly acid pH.
Ferrous leghemoglobinli complcs dots not combine with carbon monoxide2 and may be expected to bc unreactive toward oxygen as well.
Acetic acid, and other lower aliphatic mono-and dicarboxylic acids, exert profound effects on the optical spectra, magnetic properties, and chemical reactivity (16, 17)" of ferric leghemoglobin, but are without effect on the optical spectrum of the ferrous protein.
A final objective of the present study was to discover if acetic acid exerts an effect OII the chemical reactivity of the ferroprotein.

Preparation
of Leghemoglobin-Lincoln strain soybeans were grown in a glasshouse at 25-20" in a sand-vermiculite mixture inoculated with Rhizobiumjaponicum strain 505 (Wisconsin) and watered twice daily, with the addition of LIcKnight's salt solution (18) twice weekly.
Root noduIes were harvested from 5week-old plants, the leghemoglobins were extracted from them by grinding in air-equilibrated 0.1 M potassium phosphate buffer (pH 5.5 or 6.8), then centrifuging and fractionating with ammonium sulfate as described previously (8 Conditions-Experiments were performed in 0.05 M sodium pyrophosphate buffer (Na4P207 brought to pFT with HCl) at pH 5.3 and 6.8, and in 0.05 M sodium acetate builder at pH 5.3. All solutions contained 100 PM EDTA. The temperature was 20" except in the determination of the combination r:ttc constant for oxygen.
Most rnc~asurements of this rapid reaction were made at 10"; a few measurerneuts were made at 3" and the value expected at 20" estimated by graphical extrapolatioil. Apparatus-static spectra were determined in a Gary i nodd 11 recording spectrophotometer.
Reaction kinetics were measured in a Gibson-Mi1nt.s (19) stopped flow apparatus with a 2-cm light path in the obsrr\.ation cell.
Oxygen Combination Rule Constant--.inaerobic solutioils OS initially ferric leghemoglobin (0.5 PM) were reduced by the anaerobic addition of sodiunl tlithioilite, with an amount 4 to 7 times greater than that required for reduction of the protciu. These solutions were rapidly rnixcd with solutions of 2.8 pal osygen in buffer.
The dithionite remainiilg after mixing with oxygen-containing buffer was not sufficient to consume the oxygen present.
The reaction was followed at 430 nm. The nature of the product, LbOz, was confirmed by constructing a partial kinetic difference spectrum.
Under the conditions used the rate of react.ion of the hemoglobin with oxygen is very much greater than the rate of reaction of tlithionite with oxygen, and the latter reaction did not interfere.
The excess dithionite was ultimately removed by reaction with uncombined oxygen.
If the coucentration of dithionite (iu excess of the initially ferric protein) was deliberately made greater than the final oxygen concentration, a very slow (I+ = 30 set) reduction of t,he formed oxyhemoglobin by dithionite became evident. The kinetic difference spectrum for this latter reaction is diagnostic for the conversion of LbOz to ferrcJUS Lb and iilcompatible with any reaction of thr i'clrric proteiri.
This means that the ferric I)rotein did not accumulate during t,he rapid kinetic process.
The solutions of osygen in buffer were prepared by adding small amounts of air-cquilibratcd water to anaerobic buffer. Anaerobic buffer alour:, rapidly mixed with deosyhcmoglobin solutioll, gave no absorbance change, indicating that it and t,he apparatus were in fact free from significant amounts of oxygen. I\lost measurements were made at 10". The combination rate constant expected at 20" was estimated from the determinations made at lo", using the temperature dependence of the reactioh rate presented in Fig. 1. The mean of several determinations of the rate constant at 10" is increased by the factor 1.2 to obtain the rate constant at 20".

Oxygen Dissociation
Rate Constant-The velocity constant for the dissociation of oxygen was measured by two methods, with reasonable agreement between the results obtained.
Solutions of ferric Lb (8.8 FM), were reduced titrimetrically by the stepwise addition of dithionite to a 107; excess. The solution, held in a 30-ml syringe, was deosygenated by a fine stream of helium bubbled through it; the syringe was closed with :t rubber serum stopper, and the course of the titration monitored spectrophotometrically with the syringe barrel placed in the light beam of the spectrophotometer.
These solutions were injected into a siliconized round bottom flask containing oxygen, pan = 0.8 mm Hg, in helium, so that the fluid flowed smoothly in a thin layer over the glass surface and by equilibration with the gas In this way the protein was oxygenated without significant denaturation. Solutions of oxyleghemoglobin were rapidly mixed with solutions of dithionite (6 InM) in buffer (when the final ~1-1 was 6.8) or in 1 rnM NaOH (when the final pH was 5.3), and the reaction followed at a wave length near 414 nm isosbestic for ferric and ferrous Lb. The isosbestic point was determined in the stopped flow apparatus for each protein under each set of experimental conditions. This precaution is required because solutions of Lb02 at low oxygen pressure inevitably contain some ferric protein, and the rate of reduction of the ferric protein by dithionite is commensurate with the rate of deoxygenation.
The rate of deoxygenation was essentially independent of dithionite concentration from 1.5 to 6.0 mM dithionite after mixing, and the reaction was homogeneous and first order to more than 90yG completion.
In other experiments oxyleghemoglobin solutions (8.8 PM LbOs, 1.5 PM free oxygen), were rapidly mixed with solutions of carbon monoxide in buffer, and the reaction followed at 416 nm, a wave length near the absorption maximum for LbCO. The rate was nearly independent of carbon monoxide concentration from 0.5 to 0.8 mM CO after mixing.
Rates of oxygen dissociation measured in this way were somewhat slower than those measured in the presence of dithionite, reflecting the limitation to the ratio of carbon monoxide to oxygen pressure achievable in these experiments. Ferricyanide is not suitable to measure the rate of dissociation of oxyleghemoglobin.
In the presence of a very large excess of ferricyanide LbOz is converted to the ferric state, at a rate dependent on the ferricyanide concentration and manyfold less than the rate of dissociation measured by other methods. Carbon Monoxide Combination Rate Constant-Solutions of ferrous Lb (1 PM) in buffer containing 1 mM dithionite were rapidly mixed with solutions of carbon monoxide in buffer also containing 1 InM dithionite, and the reaction followed at 416 or 432 nm.
The combination rate was directly proportional to the carbon monoxide concentration from 2.5 to 15 PM carbon monoxide after mixing.

AND DISCUSSION
The rates of the several reactions are collected in Table I. Perhaps the most striking feature of the results is the very great rate of combination of leghemoglobin with oxygen. The rate constant of this reaction is greater than that of any other hemoglobin.
The kinetics of the reactions of leghemoglobin are compared with those of other hemoglobins in Table II. On the other hand, oxygen dissociates from leghemoglobin at a moderate rate, which in fact is only about one-half that at which oxygen dissociates from myoglobin ,  Table  II.
The exceptionally high affinity of leghemoglobin for oxygen, therefore, is largely a consequence of very rapid combination with oxygen. The affinity of leghemoglobin for oxygen may be calculated by combining the kinetic constants of the forward and reverse reactions.
The affinities so calculated for leghemoglobins a and c agree within a factor of two with the oxygen affinity determined directly, Table III.
The combination of leghemoglobin with carbon monoxide is also very fast, being 20 times that of myoglobin (Table II). The great affinity of leghemoglobin for carbon monoxide (3) reflects the rapid rate of combination.
ils has been noted before (27), the relative rates of combination with oxygen and carbon monoxide are similar for all the proteins examined, in the face of a 2000-fold difference between the largest and smallest combination rate constants (Table II). The two major components, leghemoglobins a and c, differ in molecular size (9), amino acid composition (29), and other properties.
The data presented here show that there are no large differences in the kinetic constants of the reactions of these two proteins with oxygen and carbon monoxide. We note that leghemoglobin c does combine somewhat more slowly with osygen than does leghemoglobin a, and that the lesser oxygen affinity of leghemoglobin c may be ascribed to this difference (Table III). The rate constants for oxygen dissociation (and for carbon monoxide dissociation) are consistently somewhat greater at pH 6.8 than at pH 5.3. The differences, although not large, exceed the probable experimental error. No differences were noted in the rate of oxygen dissociation from oxyleghemoglobin isolated as such or regenerated from ferrileghemoglobin.
It is of interest to compare leghemoglobin with animal hemoglobins and as well with oxygen-reactive plant and yeast hemeproteins (Table II).
In a general way the properties of leghemo-   ferrous leghemoglobin is unaffected by the presence of acetate (8) and there is no available criterion to show whether or not a complex is formed.
The rates of reaction of ferrous leghemoglobin a and ferrous leghemoglobin c are not affected by the presence of acetate, as shown by comparison of the results in acetate and pyrophosphate buffers at pH 5.3 (Table I). Leghemoglobin unquestionably plays an important role in the economy of the root nodule, in which it is found in abundance. Virtanen et al. (13), Keilin and Smith (4,30), and Wilson (31), were among the first to point out the obligatory relation between the occurrence of leghemoglobin and the ability to fix nitrogen. Lind and Wilson (32) have shown that nitrogen fixation in clover nodules is inhibited by CO concentrations about one-tenth those required to inhibit fixation in aerobic free living bacteria such as Azotobacter.
This inhibition is often ascribed (33) to a specific effect on nodule leghemoglobin.
Nodules respire vigorously at a rate which is diffusion limited (5), and the Rhizobium bacteroids are responsible for much of this respiration (34). The uptake of oxygen by thin slices of nodules is halved under conditions of low carbon monoxide pressure where the leghemoglobin is bound in combination with carbon monoxide (35). These facts lead us to enquire whether leghemoglobin, by facilitating oxygen diffusion, may aid the entry of oxygen into the nodule.
The ability of leghemoglobin to facilitate oxygen diffusion has not been tested experimentally.
However a protein with similar kinetics and equilibria of oxygen binding, hemoglobin I-1 ( globin resemble those of the animal hemoglobins and myoglobins and differ from those of the two peroxidases. Elsewhere2 we show that the complex formed between leghemoglobin a and the small molecule X, when ferrous, does not react with carbon monoxide, and by analogy probably is also inert toward combination with oxygen. The ferrous complex, formed rapidly by reaction with dithionite, relaxes within a few minutes to a mixture in which free ferrous leghemoglobin predominates. In the samples used here the fraction of the latter was more than 70 5;; . The reactions whose rates are reported in Table I  where the same (38).
A glut of oxygen at the periphery and a shortage in the center will be avoided.
The properties of leghemoglobin are ideally suited to favor the facilitation of oxygen diffusion in an environment of very low mean oxygen pressure.
Both combination and dissociation rate constants are adapted to this end. The requirements are that the protein be largely saturated with oxygen at the high pressure boundary of the system, near the periphery of the plant cell or root nodule.
Simultaneously it must also be largely desaturated at the low pressure boundary; for in the absence of a gradient of oxygenation, the facilitated flux vanishes.
Appleby (14), taking advantage of leghemoglobin as an internal indicator of oxygen pressure, estimates that the mean oxygen pressure within a nodule is of the order of 0.01 mm Hg, which is a very low pressure. It is, however, sufficient to support a vigorous oxygen uptake by the Rhizobium bacteroids.3 The rate constant for the combination of leghemoglobin with oxygen is the largest encountered in any hemoglobin; saturation of leghemoglobin at the periphery of the nodule is thereby assured.
The dissociation rate constant, as already noted, is sufficiently large to assure rapid unloading of oxygen at the low pressure boundary of the system, the bacteroids, and also to assure a sufficient turnover rate of oxyleghemoglobin within the bulk of the cytoplasm. The mean, volume-averaged, fractional saturation of leghemoglobin within a working nodule is about 2Ooi, (14).
It is interesting that this is of the same order as the volume-averaged saturation of myoglobin in an operating mammalian heart or red muscle (12). At steady state, the rate of oxygen combination at any point with the cell is equal to the rate of oxygen dissociation.
The mean turnover rate (known from the mean fractional saturation, hemeprotein concentration, and the dissociation rate constant), interestingly, is about the same in the root nodule as in mammalian red muscle.
Acknowledgments-Part of this work was performed at the Division of Plant Industry, C.S.I.R.O., Canberra, Australia. Soybeans were grown, nodules harvested, and some earl)-steps in the extraction and purification of leghemoglobin were done there.
We thank Mrs. L. Grinvalds for her help in this I)hnse of the work.
We thank Dr. Robert W. Yoblc for his helpiul discussions.