Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete

Frankia strain CcI3 grown in culture produced a hemoglobin whichhad optical absorption bands typical of a hemoglobin and a molecularmass of 14.1 kDa. Its equilibrium oxygen binding constant was274 nM, the oxygen dissociation rate constant was 56 s^-1, andthe oxygen association rate constant was 206 µM^-1 s^-1.

INTRODUCTION

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Introduction

Frankia, a nitrogen-fixing actinomycete, forms root noduleswith plants belonging to eight different plant families. Theseactinorhizal root nodules are modified lateral roots and containvarious amounts of hemoglobin ranging from very low concentrationsin Purshia to high concentrations in Casuarina and Myrica (18,24). In Casuarina, the hemoglobin (or at least most of it) isproduced by the plant (6, 12), but the source in other actinorhizalnodules is not known. In nodules from Alnus, which contain intermediateconcentrations of hemoglobin, much of the hemoglobin is associatedwith nodule fractions that contain Frankia (20). Based on this,we hypothesized that Frankia can produce hemoglobin.

Recently a number of truncated hemoglobins that have amino acidsequences that are 20 to 40 residues shorter than classicalhemoglobins have been described. They are found in diverse microorganisms,namely, the prokaryotes Synechocystis (8), Mycobacterium (9),and Nostoc (16), and the eukaryotes Paramecium (11), Tetrahymena(14), and Chlamydomonas (7). The amino acid sequences of thesehemoglobins indicate that they have a common evolutionary originand are only distantly related to classical hemoglobins (21).In this paper we report that Frankia produces a hemoglobin thatmay belong to this group.

Cell culture and hemoglobin extraction.

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Cell culture and hemoglobin...

Cultures of Frankia strain CcI3, originally isolated from rootnodules of Casuarina cunninghamiana (27), were grown on a definednitrogen-free medium (3) with propionate as the carbon source.Growth was in 1-liter flasks without shaking, for a period of1 to 3 months.

For hemoglobin extraction, the hyphal mat was collected on apolycarbonate membrane filter with 0.4-µm pores, and approximately1 g of moist hyphae was placed in a 15-ml glass centrifuge tube(Corex no. 8441). Then 1.0 ml of extraction buffer (0.1 M potassiumphosphate, 0.13 mM EDTA [pH 7.4]) and 2.0 g of 0.1-mm-diameterglass beads (Biospec Products, Bartlesville, Okla.) were added,and the tube was stoppered and equilibrated with carbon monoxide.After 10 min of incubation at room temperature (to help depleteany remaining O₂), the hyphae were disrupted by agitating thetube with a vortex-type tube mixer (23). Agitation was at roomtemperature for a total of 5 to 10 min, with cycles of 30 to60 s of agitation followed by 10 s of rest. After centrifugation(10 min at 4,500 x g) the supernatant was collected, and thepellet was reextracted after the addition of 1.0 ml of extractionbuffer.

Purification and molecular mass determination.

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Purification and Molecular mass...

The crude extract was purified by gel filtration at room temperatureon a 1.1- by 40-cm column of Sephacryl S-200, using the sameCO-equilibrated buffer that was used for extraction. The samecolumn was used to estimate molecular mass, with cytochromec and myoglobin from horse skeletal muscle used as standards.The hemoglobin from the gel filtration fractions was studiedwithout further purification.

The molecular mass as determined by gel filtration was 14.1± 0.1 kDa (mean ± standard error [SE]; n = 3).This is consistent with the molecular masses of 13.8 and 15kDa determined by gel filtration for the truncated hemoglobinsfrom Nostoc (16) and Synechocystis (8).

Determination of optical absorption spectra and the O₂ equilibrium constant.

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Determination of optical...

Gel filtration fractions containing carboxyhemoglobin (HbCO)were placed in a tonometer, an enzymatic reduction system (13)was added to maintain the heme iron in the ferrous form, andabsorption spectra for HbCO were taken immediately. Carbon monoxidewas displaced from the heme iron by competition with oxygenin the presence of light, followed by removal of the displacedCO under vacuum. Three cycles of this treatment converted theferrous-CO form of the protein to the oxyferrous form as judgedby the Soret and visible spectra. Next, the oxygen was removedfrom the protein by repeated flushing with pure N₂, and thespectrum of the resulting deoxyhemoglobin was taken. Followingthis, the oxygen equilibrium constant was estimated by the tonometricmethod (17), which involved stepwise addition of measured amountsof oxygen. And lastly, spectra for oxyhemoglobin (HbO₂) weretaken when addition of more O₂ showed no further increase inthe HbO₂ absorption peaks.

While the carbonmonoxy form of the hemoglobin extracted fromFrankia was very stable, the oxy and deoxy forms were not. Measurableloss of absorbance was observed over a time period of minutesat room temperature (approximately 25°C). However, additionof the enzymatic reducing system eliminated this problem, allowingmeasurements of oxygen equilibrium and dissociation rate. Theabsorption spectra observed for the carbonmonoxy, oxy, and deoxyforms of the hemoglobin (Fig. 1) are similar to those foundfor other hemoglobins, including the truncated hemoglobins fromNostoc (22) and Paramecium (11).

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FIG. 1. Optical spectra of the carbonmonoxy (open circles), oxy (triangles), and deoxy (filled circles) forms of Frankia hemoglobin. Data were taken at 1-nm intervals, and symbols were placed at 10-nm intervals. The spectra between 475 and 700 nm are expanded fivefold and vertically displaced from each other to provide clarity.

The oxygen affinity (K_d) of Frankia hemoglobin is in the rangereported for hemoglobins believed to function in O₂ transport(Table 1). However, its affinity is about 10 times less thanthose of the hemoglobins from nitrogen-fixing root nodules oflegumes and the nonlegumes Parasponia and Casuarina (25). TheHill coefficient for Frankia hemoglobin is not significantlydifferent from 1; thus, oxygen binding is not cooperative. Thisis consistent with the molecular mass determination by gel filtration,which indicates a mass equal to that of a truncated hemoglobinmonomer.

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TABLE 1. Kinetic constants for O₂ binding by hemoglobins from Frankia and other organisms

Oxygen dissociation and association rates.

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Oxygen dissociation and...

Oxygen dissociation rate constants (k_off) were measured by anApplied Photophysics SF-17 microvolume stopped-flow spectrophotometeras previously described (5). The same Frankia hemoglobin sampleused to estimate the oxygen binding constant was used in thekinetic measurements. Previous studies (5) have shown that thepresence of the components of the enzymatic reduction systemdo not affect the determination. The Frankia hemoglobin solutionswere deoxygenated by mixing with a 2.0-mg/ml solution of sodiumdithionite. Deoxygenation kinetics were followed at 430 nm.Rate constants were estimated by standard least-squares fittingof the data to a first-order mechanism. Each reported valuerepresents the average of four to eight individual determinations.The oxygen association rate constant (k'_on) was estimated fromthe equilibrium binding constant and k_off.

The oxygen dissociation rate (k_off) (Table 1) is similar tothat observed for Nostoc hemoglobin and is faster than thatof many other hemoglobins. The Arrhenius plot of these datais linear between 10 and 25°C and gives an activation energyfor dissociation of approximately 18 kcal/mol (Fig. 2), a valueconsistent with estimates for other monomeric hemoglobins (2).

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FIG. 2. Arrhenius plot of the oxygen dissociation rate (k_off) of Frankia hemoglobin. Values are means ± SE for four to eight individual determinations.

The oxygen association rate (k'_on), calculated from the k_offand the oxygen affinity of Frankia hemoglobin (Table 1), isalso similar to that measured for Nostoc hemoglobin. It is notablethat the k_off for Nostoc hemoglobin is the fastest that hasever been measured.

Effect of combined nitrogen.

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Effect of combined nitrogen.

Cultures of Frankia strain CcI3 were grown on nitrogen-freemedium as before or on the same medium supplemented with NH₄Cl(5 mM). The cultures were grown for 28 days, and the hemoglobinconcentration in the crude extract was estimated from the HbCOabsorption peak at 420 nm by the method of Tjepkema and Asa(24). This method assumes a cell density of 1.0 and convertsmass to volume. It underestimates the hemoglobin concentrationbecause not all of the cell mass is cytoplasm. On a fresh massbasis, cultures grown in medium lacking N contained 22.8 ±1.13 (mean ± SE) µM hemoglobin whereas those grownon medium with N contained only 8.22 ± 1.12 µMhemoglobin.

Our finding that Frankia can produce a hemoglobin in culturestrongly supports an earlier suggestion (20) that Frankia producesat least part of the hemoglobin in Alnus nodules. This is furthersupported by the finding that Frankia strain ArI3, originallyisolated from Alnus rubra, also produces a hemoglobin (4). Frankiais also likely to be the source of part of the hemoglobin observedin other actinorhizal nodules. In previous work, evidence wasfound for two hemoglobins in the actinorhizal nodules of Myricagale (15). When the hemoglobin from these nodules was purifiedby gel filtration, much of it eluted in a fraction correspondingto a molecular mass of 38.5 kDa, with an HbCO absorption peakat 416 nm. A smaller amount eluted in a fraction indicatinga molecular mass of 16.7 kDa, with an absorption peak at 419nm (15). Thus, it is possible that the 38.5-kDa hemoglobin wasa dimer produced by the host plant and the 16.7-kDa hemoglobinwas a monomer produced by Frankia.

The hemoglobins produced by Frankia and Nostoc have high valuesfor both k'_on and k_off (Table 1). Such values would be wellsuited for the facilitation of oxygen diffusion over short distances.Whether the concentration of hemoglobin in Frankia is sufficientfor simple facilitation of oxygen diffusion is not clear. Anoverall concentration of 23 µM was found for culturesgrown in the absence of combined nitrogen, i.e., under nitrogen-fixingconditions, and such a low concentration may not be sufficientfor significant facilitation of O₂ diffusion (26). But if muchof the hemoglobin is concentrated in specific regions of thecell such as the area near the plasma membrane, then the concentrationwould be in the range expected for facilitation of oxygen diffusion.

The oxygen binding properties of the hemoglobins from Frankiaand Nostoc contrast sharply with those of most other hemoglobins,including truncated hemoglobins other than those from Nostoc(Table 1). Evidence from Raman spectroscopy suggests an enzymatic,rather than O₂ transport, function for the truncated hemoglobinsfrom Synechocystis and Chlamydomonas (10). On the other handit is interesting that the hemoglobin from the hemolymph ofthe larvae of Chironomus, an insect, has oxygen associationand dissociation rates similar to those of the hemoglobins fromFrankia and Nostoc (Table 1). The hemoglobins from certain otherinvertebrates also have similar properties (25).

In summary, we find that cultures of Frankia produce a hemoglobinwith a molecular mass and oxygen binding properties that aresimilar to those of the hemoglobin produced by Nostoc commune.It is probable that the hemoglobin produced by Frankia in cultureis also produced in its nitrogen-fixing symbioses with higherplants.

ACKNOWLEDGMENTS

This work was supported by a University of Maine Faculty ResearchGrant to J.D.T., NRICGP/USDA grant 97-35503-4927 to R.E.C.,and CSREES/USDA grant ME08465 to C.R.S.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, 5722 Deering Hall, Orono, ME 04469-5722. Phone: (207) 581-2993. Fax: (207) 581-2969. E-mail: tjepkema{at}maine.edu.

Maine Agricultural and Forest Experiment Station external publicationnumber 2508.

REFERENCES

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