IRON IN THE WHOLE BACTERIAL CELL. EXPLORATORY INVESTIGATIONS

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IRON IN THE WHOLE BACTERIAL CELL.

EXPLORATORY INVESTIGATIONS

E. Bauminger, S. Cohen, E. Giberman, I. Nowik, S. Ofer, J. Yariv, M.

Werber, M. Mevarech

To cite this version:

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IRON IN THE WHOLE BACTERIAL CELL. EXPLORATORY INVESTIGATIONS

E. R. BAUMINGER, S. G. COHEN, E. GIBERMAN, I. NOWIK and S. OFER Racah Institute of Physics, The Hebrew University, Jerusalem, Israel

J. YARIV

Department of Biophysics, The Weizmann Institute of Science, Rehovoth, Israel and

M. M. WERBER and M. MEVARECH

Polymer Department, The Weizmann Institute of Science, Rehovoth, Israel

Résumé. — Des mesures Môssbauer ont été effectuées sur des échantillons compacts de cellules

de E. Coli et de Halobacterium de la mer morte dans des états congelés et non congelés. Les bactéries ont été cultivées dans un milieu contenant du Fe57 hautement enrichi de concentration diverses.

La forme du spectre observée pour les échantillons congelés ne dépend ni de la concentration de Fe (dans de large limites) ni des conditions de culture des bactéries. Des spectres remarquables ont été observés pour les cellules non congelées. Les spectres montrent un grand élargissement motionel (jusqu'à 5 cm/s). Les spectres observés pour les Halobacterium congelées sont comparés à celui obtenu pour le 2 Fe ferredoxin isolé de ces bactéries.

Abstract. — Recoil-free measurements were carried out on samples of packed cells of E. Coli and Halobacterium from the Dead Sea in both the frozen and unfrozen state. The bacteria were grown in media supplemented with highly enriched Fe57 at various concentrations. The shape of

the spectrum observed in the frozen samples was independent of iron concentrations over a wide range and other conditions of bacterial growth. Remarkable spectra have been observed in the unfrozen cells. The spectra display large motional broadening (up to 5 cm/s). The spectrum observed in frozen Halobacterium is compared with that obtaining in 2 Fe ferredoxin isolated from those bacteria.

1. Introduction. — Most of the Mossbauer investi-gations on biological materials have been carried out

on isolated iron-containing proteins and other compounds in vitro. These investigations have been most fruitful. However, they have been carried out in conditions which in general differ from those obtaining in the actual biological cell. A primary motivation for the present work was to explore the possibilities of investigating iron proteins through the techniques of recoil-free spectroscopy in the whole bacterial cell and to compare the recoil-free spectra with those obtaining in the isolated materials. A ferredoxin (containing 2 Fe atoms per molecule) has been isolated and identified in each of the two bacterial species investigated here,

E. Coli [1] and Halobacterium [2]. There has recently

been an increase in the number of membrane-bound Fe-S proteins known to exist in animal plant and bacterial transport systems [3]. It was therefore hoped that one might be able to identify at least iron-sulphur proteins in the whole bacterial cells using Mossbauer spectroscopy.

Some reports of earlier work on frozen bacteria have already been published [4, 5] by Moshkovski et

al. In the present work measurements of the recoil-free

absorption of Fe5 7 were carried out in both frozen and

non-frozen bacterial cells. A special effort was made to look for recoil-free effects in non-frozen bacterial cells. Any such effects must be ascribed to iron bound in a quasi-solid environment. It was thus hoped that a comparison between spectra obtaining in frozen and non-frozen cells, might yield valuable and possibly rather unique information on the physical states and various sites of iron-proteins and compounds in the whole cells. Of particular interest and importance might be the identification of iron sites which are membrane bound. Recoil-free effects in both frozen and unfrozen cells of E. Coli containing 57

Co-entero-chelin (enterobactin) have been reported in a previous communication [6].

Measurements were carried out on samples of packed cells of E. Coli and Halobacterium from the Dead Sea. The latter bacterium lives in a highly saline environment. Two strains of E. coli were used : The 3 300-K12 strain and the haemin-less mutant H7

(cyto-chrome-free) [7]. The bacteria were grown in media supplemented with highly enriched Fe5 7.

The measurements on frozen cells were carried out at various temperatures, between 4 K and the freezing

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C6-228 E. R. BAUMINGER et al. temperature. Most of the experiments with unfrozen

cells were carried out at 3 OC. A few measurements

were also made at higher temperatures.

Two further sets of measurements were made using the Halobacteria grown in media enriched in Fe5, :

a) The recoil-free spectra of an isolated 2 Fe ferredoxin were measured. This data is compared to the spectra obtaining in the whole cells.

b) The recoil-free spectra of membrane fragments were examined in frozen suspension after fragmenta- tion by osmotic shock.

2. The Preparation of Microbiological and Biochemi- cal Samples.

-

2.1 E. COLI. - Both strains were grown in glassware and media treated for removal of iron and supplemented with FeSO,, 90

% enriched

with Fe5,. The 3 300-K 12 strains were grown aero- bically in medium-56 with succinate as the carbon source and the anaerobic H, strain (cytochrome free) with medium-56 supplemented with 0.3

%

casamino acids and glucose as the carbon source. Cells were grown at 37 OC and harvested, washed and sedi- mented, kept as a paste at 4 OC and transferred for measurement to a perspex sample holder. The concentration of bacteria was

-

1012 bacterialml in the final samples. In the case of the K 12 strain, Fe5, over

a very wide range of concentrations could be inclu-

ded in the samples (between

-

10 pg Fe57 per gram bacteria and

-

1 mg Fe5,lgrarn bacteria). In the case of the haeminless mutant the upper limit was much smaller (- 60 pg Fe5, per gram bacteria). It is diffi- cult to carry out Mossbauer measurements on samples (vol

-

0.5 ml) containing less than

-

5 pg FeS7/ gram bacteria.

2.2 HALOBACTERIA. - 1) Halobacteria from the Dead Sea (not so dead !) were grown as described [2], except that 1 mg/l of 57Fe in the form of FeS0,.7 H,O was added to the medium. The cells were harvested and washed twice with a solution of 4.3 M NaCl containing 0.01 M Na-phosphate buffer of pH 7.3. The samples of packed cells were then prepared as described above for E. Coli.

2) Preparation of membrane fragments and isolated ferredoxin : The first precipitate after sonication and centrifugation [8] was termed membranes and was washed as above. Ferredoxin was isolated from the supernatant of the sonicate and purified according to a procedure to be published [2]. The identification of the 2 Fe-ferredoxin is based on the following facts [2] :

The molecular weight of this protein as determined by sedimentation equilibrium was 17.500 daltons, containing

-

2 g atoms of iron per gram-molecule ; the protein contained inorganic sulphide. The visible absorption spectrum displayed maxima at 277, 330, 420 and 465 nm. The ratio of absorbancies between 420 and 277 nm was 0.32. The bacteria usually contained about 1-2 mg of ferredoxin per gram of wet bacteria.

The isolated oxidised ferredoxin was kept in 2 N aqueous NaC1. Reduced ferredoxin was prepared using dithionite and preserved in an oxygen-free environment.

3. Technique of Recoil-Free Measurements. -Under

the conditions obtaining in some of the experiments (using non-frozen bacteria or low Fe5, concentration in frozen bacteria) it was necessary to measure very small recoil-free effects (- 0.3

%).

It was therefore important to maximalise the counting rates in the resonance absorption measurement. To this end a 100 mC CoS7

-

in Rh source, and a Harwell propor- tional counter, running at about lo5 total countsjsec were used (I).

4. Recoil-Free Experiments.

-

4.1 HALOBACTERIUM

AND SEPARATED 2 Fe-FERREDOXIN.

-

4.1 .1 Isolated

Ferredoxin. - The resonant absorption spectrum of the isolated 2 Fe-ferredoxin in the oxidised state kept in saline 2 M NaCl solution was measured at 4 K, 82 K and 223 K. Figure 1 shows the spectrum obtained

-2.0 -1.0 0.0 1.0 2.0

VELOCITY ( m n l s )

FIG. 1. -Recoilless absorption spectrum of the 14.4 keV gamma ray of Fes7 in isolated 2 Fe-ferredoxin (in the oxidised state) from Halobacteria at 82 K. The solid line is the least squares computer fit to the spectrum. It is the sum of two quadrupole subspectra (dashed lines) of equal intensity with

parameters given in the text.

at 82 K. The spectrum is in a general way similar to that of other 2 Fe-ferredoxins, but shows a rather more definite resolution into two non-equivalent sites of equal intensity, each site characterized by a quadrupole doublet. Computer analysis of the data gives an excel- lent fit with the following values for quadrupole splittings and isomer shift at the two sites at 82 K

-

eq,Q/4 = (0.240 +_ 0.003) mm/s, IS, (isomer shift relative to metallic Fe) = 0.232(5) mm/s, eq,Q/4= (0.434+ 0.003) mm/s and IS, = 0.272(5) mm/s. The width at half maximum height of each line is 0.25 mm/s.

The spectrum at 4.1 K is similar and shows no evidence of any magnetic splitting.

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The spectrum of a reduced sample of the isolated ferredoxin was also measured and showed that half the iron in the protein, corresponding to site (1) in oxidized ferredoxin, is present in the form of a ferrous quadrupole doublet, as characteristic of the spectra of other reduced 2 Fe ferredoxins [9]. However, these spectra were not completely clean and indicated possibly some denaturation of the protein.

4.1.2 Frozen Halobacteria.

-

Figure 2 shows the resonant absorption spectrum obtaining at 82 K from a sample of frozen bacteria containing about 10 pgm Fe57 per gm of bacteria. This spectrum is clearly distinct from that of the isolated 2 Fe-ferre-

VELOCITY (mn/s 1

FIG. 2. -Recoilless absorption spectrum of the 14.4 keV gamma ray of Fe57 in a frozen sample of Halobacteria at 82 K . Absorber contained about 300 mgm/cm2 packed cells (with

10 pg Fes7fgrm of cells).

doxin. Rough estimates based on the biochemical yields of the isolated 2 Fe ferredoxin suggest that the latter represents about 10 % of the total iron containing proteins and other molecules in the bacterial samples. The width and shape of the lines in figure 2 suggests a spectrum of complex origin. A good computer fit can be obtained assuming a superposition of two quadrupole doublets of equal intensity. Howe- ver, the line-width of a single quadrupole line is then 0.43 mm/s, considerably greater than a line width of 0.25 mm/s obtaining with the resolved spectra of the isolated 2 Fe-ferredoxin, suggesting a complex situa- tion - either a number of components or the presence of line-broadening factors.

4.1 . 3 << Membranes >>. - The absorption spectrum of the membrane fragments precipitated after sonication of the bacteria were measured at 82 K. The spectrum is essentially identical with that obtaining in the frozen bacteria, indicating that the major part of the iron-containing components are retained with the membranes and are probably membrane bound.

4.1 .4 Unfrozen Halobacteria.

-

So far we have not been able to introduce sufficiently large quantities of Fe57 into the bacteria during growth as would make possible significant estimates of any recoil-free effects in unfrozen bacteria.

4.2 E. COLI. - 4.2.1 Frozen Bacteria. - The resonance absorption of Fe87 in frozen samples of

packed E. Coli cells were measured many times at different temperatures above 4 K and using samples prepared under different conditions, corresponding to the two different mutant strains at our disposal, variation of Fe57 concentration over a large range (e. g. at 10 pg and at 1 mg Fe5' per gram bacteria in

K 12 strain-aerobic growth), aerobic and unaerobic growth.

A remarkable result of the present investigation is

that the shape of the resonance spectra obtained was identical in all these various experiments, despite the very different conditions of growth and iron concentrations. Two typical spectra are displayed in figure 3.

-4 - 3 -2 -1 0 1 2 3 3 VELOCITY (mnls)

FIG. 3. - Recoilless absorption spectra of the 14.4 keV gamma ray of Fe57 in a) 3 300

-

K 12 E. Coli whole cells at 223 K. The absorber contained

-

400 mgIcm2 packed cells (with 400 pg Fe57/grm cells) ; b) H7 E. Coli whole cells at 223 K. The absorber contained 400 mg/cm2 packed cells (with 60 pg Fe57/grm cells). The solid line in figure b) is the sum of the quadrupole sub-spectra (dashed lines) with parameters given in

the text.

The same spectrum was observed at the very lowest iron concentration

-

--

10 pg Fe57/1 gm bacteria. The spectral shape obtaining in frozen E. Coli is very similar, although not identical, within experimental error, to that obtaining in frozen Halobacterium (Fig. 2) (and in the Halobacterium membrane fragments). This, of course, suggests a common origin in similar iron protein or iron-containing compounds. Again here, the experimental spectrum can be broken down, formally, into two quadrupole doublets of equal intensity with wide line-width

(at 223 K : eqlQ/4 = 0.239(3) mm/s ;

The presence of a relatively small fraction of reduced spectrum containing ferrous-like quadrupole spectra could usually be discerned. This amount of reduced fraction depends on the history of the samples and could be increased, for example, by : a) leaving bacteria for appreciable periods of time above 0 OC ; b) artificial reduction using dithionite.

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C6-230 E. R. BAUMINGER et al.

concentration in the nutrient media over a wide range. The upper limit of the concentration of Fe57 in the bacterial samples (as indicated by the intensity of the spectra in the frozen samples) was much greater for the

K 12 strain of E. Coli (under aerobic growth) than for the cytochrome free mutant (- 1 mg Fe5'/grm bacteria as compared to

-

60 pg Fe57/grm) and much greater in aerobic growth than in unaerobic growth of the

K 12 strain. Moreover, much less iron could be introduced in Halobacteria than in E. Coli.

4.2.2 Unfrozen E. Coli. - Most of the recoil-free measurements were taken at 3 OC. In each case two identically equal samples of the packed cells were prepared. The absorption in one of the two samples was measured (usually over a period of several days a t 3 OC) immediately after preparation. The other sample was kept frozen at liquid nitrogen temperature until the spectra at low temperatures could be measured. Thus, for a given type of growth, data on both frozen and unfrozen cells were obtained. Appreciable recoil-free effects were observed in all samples. The spectra obtained are very remarkable in that : a) the spectra are very wide (up to a few cms/s) indicating large motional broadening ; b) two components (narrow and wide) are visible. Some typical spectra are displayed in figure 4. The latter features are common to all spectra corresponding to different conditions of bacterial growth, although the spectra differ in details such as exact widths and ratio of narrow to wide components. The total intensity (integrated area) of the spectra taken at 3 OC were an appreciable fraction of the integrated area of the spectrum obtaining in the corresponding frozen samples at low temperatures. This suggests, that the iron component giving rise to the wide spectra at 3 OC is a major component of the iron-complexes respon- sible for the spectra observed in the frozen bacteria. It was assumed, in the analysis of the motional broadening spectra obtained at 3 OC, that the total

spectrum could be represented by the sum of two

-2.0 -1.0 0.0 1.0 2.0 3.0

VELOCITY (cm/s)

FIG. 4. - Recoilless absorption spectra of the 14.4 keV gamma ray of Fe57 in a) super clean (after several washings) 3 300 - K 12 E. Coli whole cells at 3 OC. The absorber contained about 800 mglcm2 packed cells with

--

1 mg Fe57lgrm cells ;

b) 3 300 - K 12 E. Coli whole cells at 3 "C. The absorber

contained about 800 mg/cm2 packed with 500 pg Fe57/grm cells ; c) H7 separate E, CoU whole cells at 3 OC. The absorber contained about 800 mg/cmz packed cells with

-

60 pg Fe57lgrm cells. The solid lines are theoretical fits to the experimental spectra, assuming motional broadening of the static spectra

obtaining at low temperatures. Each solid line is the sum of two Lorentzian broadened spectra with parameters and inten-

sities given in table I.

Lorentzian broadened spectra of different widths and intensities, each subpectrum corresponding to a motional broadening of the original static spectrum as observed at low temperatures in the frozen E. Coli. The results of such a breakdown and computer analysis are shown in figure 4 and table I. The latter displays the widths

rw

and

TN

produced by motional

Wide and narrow components in recoil-free spectra obtained at 3 OC in various unfrozen bacterial samples of E. Coli

Approximate Iron Ratio Jump Frequency

Concentration Width-mms/s Intensity ow W N A (3 OC)(*)

Bacterial Samples ~ g F e ~ ~ / g r bacteria Tw

r~

IN/Iw

108/s 107/s A(- 50 OC)

-

K 12 strain-aerobic

growth 500 26(2) 1.2(2) O.lO(1) 9 4 0.10

K 12 strain aerobic- growth super clean

preparation 1 000 12(1) 1.0(2) 0.24(2) 4 4 0.15

HM strain 60 12(2) 1.8(6) 0.31(15) 4 6 0.23

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broadening in the wide and narrow components, components giving rise to the recoil-free effects respectively, and also the ratio of the corresponding observed here. In such biochemical work Mossbauer intensities IN/Iw for several bacterial samples. spectroscopy could be advantageously used as a tracer technique. Until such a separation and identification is

5. Discussion and Conclusions.

-

5.1 A 2 Fe-ferre-

doxin from Halobacterium has been separated and identified. The recoil-free spectra of the isolated protein in frozen solution has been measured and analyzed. The main iron-components in the whole cell give rise to a spectrum in frozen cells which differs from that of

made, a discussion of the biological significance of our results is somewhat premature. Nevertheless, we shall speculate on the assumption that the unknown components are iron-proteins. On this hypothesis we list and comment on some alternative explanations for the motional broadening spectra at 3 O C .

the isolated ferredoxin. The 2 Fe-ferredoxin represents a) The broadening is produced by the Brownian only a small part of the total iron complex in the cell. _{motion of the whole cell. Rough estimates taking into} 5.2. The similarity between the spectrum obtained account the size of the individual cells and the high in Halobacteria and E. CoIi with the frozen bacteria viscosity of the packed cells suggest this contribution to suggest that the origin of both spectra is in a type of the broadening should be rather small and cannot iron-protein or compound common to both. In Halo- contribute to the wide components. Moreover, on bacterium the component seems to be membrane this hypothesis it is hard to explain the co-existence of

bound. both a wide and a narrow component.

5.3. The mot~onally broadened spectra at 3 O C is

produced by a substantial fraction of the total iron- containing components in the cell.

5.4. We have been concerned lest some of the striking spectra observed in the bacterial samples may have been produced by some unusual artifact essentially external to the cells, e. g. colloidal iron precipitated with the bacteria on centrifugation. But the unusual degree of reproducibility of effects under varying conditions of growth and our complete failure to eliminate the phenomena by working under super- clean conditions give us some confidence that the effects are real, biologically speaking. Now, a great variety of iron-proteins, fulfilling many different functions, are known to exist in the bacterial cell :

These include haem-proteins, ferredoxins of various kinds (2 Fe, 4 Fe and 8 Fe) and other Fe-S-proteins,

various enzymes containing iron, including molecules of high molecular weight. Some of these components exist in solution in the cytoplasm. Some are known to be membrane bound. Very often we are ignorant of the location in the cell. Moreover, many compounds involved in iron-transport (siderochromes) are known. In order to explain the constancy of the spectral shape obtaining in the frozen bacteria under extremely varying conditions, we think it probable that the spectra is produced by either a single major component or by a number of components, occuring in a fixed ratio of intensities.

The cytochromal components (haem) which are present in strain K 12, but not in H, strain, must be only a very small fraction of the total iron component, since no discernable difference in spectra shape between the two strains has yet been observed.

5.5. It seems to us that in order to clarify the nature of the phenomena described here, it would be important to try to isolate and identify the component or

b) Motion of iron proteins in the cytoplasmic membrane.

c) Motion of iron proteins in solution in the cytoplasm. b) and c) could even be neatly combined by assuming the wide component produced by some soluble Fe-S protein in the cytoplasm and the narrow components by a less mobile Fe-S protein in the membrane.

On a simple jump model of motional broadening the maximum jump frequency is given by r / 2 R where

r

is the measured motional broadening (in units of energy). The jump frequencies found in this way ow and ow corresponding to the widths, T, and

rN

for the two components are given in table I. On the other extreme model of continuous diffusion

r

= 2 hk2D, where k is the wave-number of the gamma rays and D is the diffusion coefficient. In this way one obtains a typical value of Dw for the wide component

- --

cm2/s, and for D, (narrow component)

--

lo-'' cm2/s). On the assumption that the motion of the protein can be described by the Brownian motion of a spherical particle of radius R in a medium of viscosity q, the following relation obtains

where

rN

is the natural width and z is the- lifetime of the excited state.

Assuming, somewhat arbitrarily, R

--

20

A

we obtain, as rough estimates for the effective viscosities felt by the proteins in the two environments for case I

(of Table I) :

rw

--

7 poise, qN

--

140 poise.

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C6-232 E. R. BAUMINGER et a/.

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