LEGHEMOGLOBIN: THE ROLE OF HEMOGLOBIN IN THE NITROGEN-FIXING LEGUME ROOT NODULE*

Jonathan B . Wittenberg?

Department of Physiology Albert Einstein Coiiege of Medicine

New York, New York 10461

C . A. Appleby, F. J. Bergersen, and G. L. Turner

Division of Plant Industry Commonwealth Scientific and Industrial Organization

Canberra, A.C.T. 2601, Australia

A world increasingly short of protein may expend its limited supply of chemical or electrical energy t o fix atmospheric nitrogen into ammonia, or may rely in greater part on plants to harvest solar energy, some part of which is directed t o the support of bacterial nitrogen fixation. The rapid development of the nitrogen-fixing soybean as a major crop suggests that reliance on plants may already have become the more economical way t o regenerate the supply of fixed nitrogen. A hemeprotein, leghemoglobin, is an indispensable component of the system by which leguminous plants support the activity of nitrogen-fixing bacteria. We here inquire into the molecular mechanism by which leghemoglobin augments the oxygen consumption and nitrogen-fixing activity of bacteroids. That mechanism is leghemoglobin-facilitated oxygen diffusion brought about by translational diffusion of the oxygenated protein. The flux of oxygen is enhanced by facilitated diffusion. In addition, we now discover, facilitated diffusion makes oxygen more available to the terminal oxidases of subcellular organelles. We are concerned with the physicochemical definition of what we mean by “available.”

Nitrogen-fixing nodules are formed on the roots of legumes in response t o invasion by bacteria of the genus Rhizobium. Rhizobia, modified for symbiotic life, are called bacteroids. The enzyme complex, nitrogenase, which is responsible for nitrogen fixation, is located wholly within the bacteroids. Nitrogenase does not utilize oxygen (in fact it is inhibited by even traces of oxygen) but depends for its activity of a supply of ATP formed (presumably) by bacteroidal oxidative phosphorylation. The bacteroids, which occur within the nodule cell, are in some ways analogous to muscle mitochondria. They occupy about the same fraction, approximately one-third,1,20f the cell volume and are responsible for the largest part of the oxygen consumption. Bacteroidal oxygen demand is vigorous; the oxygen consumption of the intact nodule is about one-tenth as great as that of the most active mammalian muscles. In fact, both

* This work was supported in part by Research Grant GB 36571X to J.B.W. from the

t Research Career Program Awardee 1-H6-733 of the United States Public Health National Science Foundation.

Service, National Heart and Lung Institute.

28

Wittenberg et al. : Leghemoglobin 29

nitrogen fixation and oxygen consumption in the intact nodule are oxygen- limited and increase in response t o increased ambient oxygen pressure.

Leghemoglobin is a monomeric protein, molecular weight 16,400, carrying one protoheme IX per molecule and binding reversibly one molecule of oxygen. It is synthesized by the plant, and the genetic information for its synthesis is carried by the plant genome. The amino acid sequence has been determined3 and shows analogies to those of vertebrate myoglobins. The affinity for oxygen is very great,4 and both equilibria and kinetics of the reaction with oxygen are favorable for the facilitation of oxygen diffusion in an environment of very low oxygen p r e ~ s u r e . ~ ,6 Leghemoglobin occurs in the cytoplasm of the bacteroid- containing cells of the central tissues of the soybean root nodule at an effective concentration of about 1 mM, which is a large concentration, commensurate with the concentration of myoglobin in mammalian red muscle fibers. I t appears t o be in direct contact with the surface of the bacteroids.

A typical soybean root nodule is nearly spherical and about 1-3 mm in diameter. It consists of a homogeneous central tissue surrounded by a woody cortex. Leghemoglobin may be used as an internal indicator of the volume- averaged oxygen pressure within the cells of the central t i s ~ u e ; ~ . ~ that pressure is very small, about 0.01 mm Hg. It is maintained at this level by a vigorous oxygen consumption for which the bacteroids are mainly responsible. The influx of oxygen may be limited by diffusion through the cortex. A simple calculation shows that, given the known oxygen consumption, and the measured thickness of the cortex (150 micrometers, Reference l ) , the resistance of the cortex t o the inward flow of atmospheric oxygen required t o achieve the known internal oxygen pressure need be no greater than the resistance that would be offered by an equivalent layer of water. Within the central tissue, a recently discovered system of air passageways* probably maintains the oxygen pressure at the surface of the cells of the central tissue everywhere the same. It is interesting t o note that the density of these very fine passageways is about the same as the density of capillaries in the mammalian heart, and that the geometry of the cytoplasmic path through which oxygen must diffuse is about the same in the two tissues.* The analogy with muscle suggests that this path could offer a considerable impediment to the inward flow of oxygen.*

The oxygen uptake of intact nodules is clearly diffusion limited, but J. D. Smith,' working in Keilin's laboratory, could not show a decrease in oxygen uptake which might be expected when leghemoglobin function is abolished by carbon monoxide. Diffusion-limited oxygen consumption of thin slices of nodules, however, is inhibited partially by carbon monoxide.' " The question was resolved very recently when Bergersen, Turner and Appleby' measured simultaneously oxygen uptake and nitrogenase activity of intact nodules. Although oxygen uptake was but little affected by carbon monoxide, nitro- genase activity (measured by carbon monoxide-insensitive hydrogen evolution) was essentially abolished. In further experiments using dense suspensions of isolated bacteroids, t o which leghemoglobin could be added, they proved that the effect of carbon monoxide was t o block the action of leghemoglobin. In the experiments reported here, we have used dilute suspensions of isolated bacteroids t o explore the molecular mechanism of leghemoglobin action. A fuller account is presented elsewhere.'

We find that oxyleghemoglobin, present in well-stirred suspensions of bacteroids isolated from soybean root nodules, enhances the rates of oxygen consumption and of reduction of acetylene t o ethylene, a measure of the activity of the enzyme complex nitrogenase. The rate of incorporation of labeled

30 Annals New York Academy of Sciences

nitrogen from l S N , into N H 3 , another measure of nitrogenase activity, is also augmented. We are here concerned with the events a t the interface between the solution and the bacteroid surface.

The average of the results of our experiments is presented in TABLE 1. In the absence of oxyleghemoglobin bacteroids maintain a substantial rate of oxygen uptake, which is largely ineffective in supporting nitrogenase activity. Nitro- genase activity in the absence of leghemoglobin is small, about one-tenth the activity expressed under optimal conditions. Oxyleghemoglobin present in such a suspension brings about an increment of oxygen consumption which, under the near optimal conditions adopted, exceeds the oxygen consumption in the absence of leghemoglobin. The total oxygen uptake, increment plus ineffective oxygen uptake, is more than doubled by leghemoglobin. The large incremental oxygen uptake brought about by leghemoglobin present in the bacteroid suspension may be considered effective in supporting nitrogenase activity because it is accompanied by a commensurately large increment in the rate of acetylene reduction. The nitrogenase activity expressed in the presence of leghemoglobin and reported in TABLE 1 is near maximal for any conditions we have tested.

TABLE 1

UPTAKE AND ACETYLENE REDUCTION BY BACTEROID SUSPENSIONS*

LEGHEMOGLOBIN-AUGMENTED OXYGEN

Acetylene Reduction

(nmol min- 1 mg- 1) Oxygen Uptake C2H4 Formed

(nmol min-1 mg-1)

Leghemoglobin absent 1.7 0.43 Leghemoglobin present 16.5 6.03 Increment 8.8 5.60

* Values are the average of 16 experiments. >

In our full report we show that leghemoglobin-facilitated diffusion of oxygen across a thin layer of solution, the “unstirred layer,” surrounding each bacteroid offers a sufficient explanation of the observed leghemoglobin-augmentation of oxygen uptake. We here inquire: how is leghemoglobin-bound oxygen delivered t o the bacteroid surface? We note that facilitation of oxygen diffusion will occur whenever oxygen is removed t o a significant extent from its combination with leghemoglobin near the low pressure boundary of the system, and that it is independent of the process by which this is accomplished.

All of the proteins tested which bind oxygen reversibly were found to augment ( t o a greater or lesser extent) bo th oxygen uptake and acetylene reduction by bacteroids (TABLE 2). Among those tested were hemoglobins from organisms in six phyla and two non-hemeproteins, hemerythrin and hemocyanin. These proteins encompass a wide range of kinetics and equilibria in their reactions with oxygen. Any specific interaction between groups on the bacteroid surface and each of such diverse proteins seems highly unlikely. We conclude that oxyleghemoglobin and the other proteins tested exert their effects by moving oxygen through the solution and thereby making molecular oxygen

Wittenberg et al. : Leghemoglobin 31

TABLE 2

AUGMENT UPTAKE AND ACETYLENE REDUCTION BY BACTEROID SUSPENSIONS

SOME OXYGEN-BINDING PROTEINS WHICH

Number of Oxygen Oxygen Molecular Binding Sites Affinity,

Weight per Molecule P% (mm Hg)

Leghemoglobin a 16,400 1 0.04

Whale myoglobin 18,000 1 0.7 Aplysia myoglobin 17,000 1 2.7 pPMB chains of Hb A 16,800 1 2.65 Hb A, stripped 64,500 4 5.5 Hb A plus inositol hexaphosphate 64,500 4 30-50 Earthworm Hb 3,300,000 144 8.2 Ascaris body wall Hb 37,000 1 0.11 Ascaris perienteric Hb 328,000 8 0.002 Hemerythrin 107,000 8 2.2 Hemocyanin 455,000 6 8-10

Gastrophilus Hb 34,000 2 0.02

available a t the bacteroid surface. We shall have t o define what we mean by “available.”

The present study emphasizes that extremely low pressures of oxygen prevail at the bacteroid surface. Among the proteins which augment the oxygen uptake of bacteroids are leghemoglobin, gastrophilus hemoglobin, and ascaris perienteric hemoglobin with oxygen affinities given by py2 of 0.04, 0.02, and 0.002 mm Hg, respectively. In order for these macromolecular carriers to augment the oxygen uptake of bacteroids, the oxygen pressure at the bacteroid surface must be sufficiently low t o remove oxygen from its combination with the carriers to a significant extent. This pressure, therefore, must be of the order of 0.02 to 0.002 mm Hg.

The several current mathematical descriptions of hemoglobin-facilitated oxygen diffusion are each addressed t o the question of the oxygen flux through the whole thickness of the layer of solution. T o reach solutions they must be to some extent arbitrary in assigning precise boundary conditions. However, what we mean here by “available” oxygen at the bacteroid surface is precisely the low pressure boundary condition. Certainly it involves a stable, moderately increased oxygen pressure near the boundary.

An important aspect of the operation of any macromolecular carrier- facilitated ligand diffusion system is that, at constant flux, the concentration of the ligand, in this case oxygen, will be increased near the low pressure boundary of the system. To appreciate this effect, consider that the macromolecular carrier cannot cross the low pressure boundary of the system (in this instance the bacteroid surface). The bound oxygen flux vanishes and the total oxygen flux across the boundary is supported by the flux of free oxygen. This in turn implies that the local gradient of free oxygen concentration adjacent t o the low pressure boundary must be made steeper by the presence of oxyleghemoglobin. Our evidence shows that this change near the boundary is sensed by the bacteroid terminal oxidases which, by definition, are a t the very boundary. In a forthcoming publication’ our colleague, Dr. A. N. Stokes, presents a challeng- ing view of how this may be brought about.

32 Annals New York Academy of Sciences

The experiments described here were done under conditions where leghemo- globin brings about an increment of oxygen flux. In this condition an additional effect comes into play. We believe, without presenting formal proof, that under this condition leghemoglobin may increase the oxygen pressure at the bacteroid surface from a very small value to some larger value, of the order of the oxygen pressure required for half-saturation of the protein, 0.04 mm Hg.

To accommodate the concept of ineffective and effective oxygen uptake it is convenient to describe our findings in terms of two bacteroid terminal oxidases of differing oxygen affinity; one of high affinity responsible for that fraction of the bacteroid oxygen uptake which is ineffective in supporting nitrogenase activity, and one of lesser affinity responsible for the effective respiration. The metalloproteins implicated in electron transport in bacteroids have been investigated spectrophotometrically.' However, there is little information as t o which of these proteins may actually serve as terminal oxidases during ineffective and effective respiration. Studies addressed t o this question are underway in the laboratory of Dr. C. A. Appleby at Canberra.

We point out that the operation of more than one terminal oxidase in the cells of a tissue offers an opportunity for the control of the direction of metabolic activity by oxygen availability. In the instance of bacteroids, the extra oxygen made available by leghemoglobin-facilitated oxygen diffusion changes the respiratory pattern from ineffective oxygen uptake only t o the sum of ineffective plus effective oxygen uptake. The activity of the isolated nitrogenase enzyme system increases as the ratio ATP/ADP increases. We may speculate that ATP formed consequent t o the effective uptake of oxygen increases the ratio of ATP/ADP within the nitrogenase domain of the bacteroid and thereby brings about the dramatic switching on of nitrogenase activity.

A fuller account of these results and complete references are given elsewhere. * The chemistry, biology, and function of leghemoglobin have been r e v i e ~ e d . * ? ~ , ~

Acknowledgment

The work described was done while the senior author was the guest of the Division of Plant Industry, Commonwealth Scientific and Industrial Organiz- ation, Canberra, Australia. I thank them for their hospitality.

References

1. BERGERSEN, F. J. & D. J . GOODCHILD. 1973. The cellular location and concentration of leghemoglobin in soybean root nodules. Aust. J . Biol. Sci. 26:

2. WITTENBERG, J. B. 1970. Myoglobin-facilitated oxygen diffusion: role of rnyoglobin

3. ELLFOLK, N. 1972. Leghemoglobin, a plant hemoglobin. Endeavour 31: 139-142. 4 . APPLEBY, C. A. 1961. The oxygen equilibrium of leghemoglobin. Biochim. Biophys.

Acta 60: 226-235. 5. WITTENBERG, J . B., C. A. APPLEBY & B. A. WITTENBERG. 1972. The kinetics of

the reactions of leghemoglobin with oxygen and carbon monoxide. J. Biol. Chem.

6. IMAMURA, T., A. RIGGS & Q. H. GIBSON. 1972. Equilibria and kinetics of ligand binding by leghemoglobin from soybean root nodules. J . Biol. Chem. 247:

741-756.

in oxygen entry into muscle. Physiol. Rev. 50: 559-636.

247: 527-531.

521-526.

Wittenberg ef al : Leghemoglobin 33

7. APPLEBY, C. A. 1969. Properties of leghemoglobin in vivo, and its isolation as ferrous oxyleghemoglobin. Biochim. Biophys. Acta 188: 222-229.

8. BERGERSEN, F. J. & D. J. GOODCHILD. 1973. Aeration pathways in soybean root nodules. Aust. J. Biol. Sci. 26: 729-740.

9. SMITH, J. D. 1949. Hemoglobin and the oxygen uptake of leguminous root nodules. Biochem. J . 44: 591-598.

10. TJEPKEMA, J . D. 1971. Oxygen transport in the soybean root nodule and the function of leghemoglobin. Ph.D. thesis. University of Michigan, Ann Arbor, Michigan.

1 1 . BERGERSEN, F. J., G. L. TURNER & C. A. APPLEBY. 1973. Studies of the physiological role of leghemoglobin in soybean root nodules. Biochim. Biophys. Acta 292: 271 -282.

12. WITTENBERG, J. B., C . A. APPLEBY, F. J. BERGERSEN & G. L. TURNER. 1974. Facilitated diffusion: the role of oxyleghemoglobin in nitrogen fixation by bacteriods isolated from soybean root nodules. J. Biol. Chem. 249: 4057-4066.

13 . STOKES, A. N. 1974. Facilitated diffusion: the elasticity of oxygen supply. Submitted for publication.

14. APPLEBY, C. A. 1969. Electron transport systems of Rhizobium japonicum. I. Haemoprotein P 4 5 0 , other CO-reactive pigments, cytochromes and oxidases in bacteroids from nitrogen-fixing root nodules. Biochim. Biophys. Acta 172: 71 -87.

15. APPLEBY, C. A. 1974. Leghemoglobin. In Biological Nitrogen Fixation. A. Quispel, Ed. North Holland Publishing Co. Amsterdam, The Netherlands. In press.

Discussion

MR. SU (State University of New York, Buffalo, N . Y . ) : What happens to the level of leghemoglobin during the growth of the whole plant? For the case of nitrogenase the relative ratio of N2 to 02 gas has a pronounced effect on the vegetative and regenerative processes of the plant. Evidently the oxygen can inhibit the reduction of nitrogen by nitrogenase. DR. WITTENBERG: First, the rate of functioning of root nodules has been

known for a long time t o be dependent on the oxygen pressure surrounding the nodule. When you increase the oxygen pressure, the rate of functioning of nitrogenase goes up. There’s a limit to this because nitrogenase is either destroyed or totally inhibited by even the most minute trace of oxygen. So there is an oxygen dependence. I d o not know whether there is any dependence on nitrogen pressure; there ought to be. Nitrogen is only five times as abundant as oxygen in the atmosphere, but of course you are using several oxygens for each nitrogen reduced, so I don’t know the answer t o that.

Concerning the life history of the plant, the leghemoglobin appears just before nitrogen fixing starts, it increases t o a maximum, and then the whole thing dies away after about thirty odd days. But the leghemoglobin appears before the nitrogenase comes into action. DR. T. SPIRO (Princeton University, Princeton, N.J.): Since specific protein

interactions have been ruled out, might not the notion that it is only the oxygen pressure in the unstirred layer that counts be relatively easily established by physical changes in the layer itself? For example, can you alter the reduction of acetylene by the addition of a mixed solvent system or some physical effect on the layer?

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DR. WITTENBERG: Yes, but such experiments are not easy t o do. Organic solvents kill the bacteroids, and unless perhaps you use glycerol there are enough stirring problems already in these crude experiments. As we improve the technology and become more sophisticated we may be able t o do such experiments.