Study of the rhizobacterium Azospirillum brasilense Sp245 using Mössbauer spectroscopy with a high velocity resolution: Implication for the analysis of ferritin-like iron cores

The results of a comparative study of two samples of the rhizobacterium Azospirillum brasilense (strain Sp245) prepared in different conditions and of human liver ferritin using Mössbauer spectroscopy with a high velocity resolution demonstrated the presence of ferritin-like iron (i.e. iron similar to that found in ferritin-like proteins) in the bacterium. Mössbauer spectra of these samples were fitted in two ways: as a rough approximation using a one quadrupole doublet fit (the homogeneous iron core model) and using a superposition of quadrupole doublets (the heterogeneous iron core model). Both results demonstrated differences in the Mössbauer parameters for mammalian ferritin and for bacterial ferritin-like iron. Moreover, some differences in the Mössbauer parameters were observed between the two samples of A. brasilense Sp245 related to the differences in their preparation conditions. 2014 Elsevier B.V. All rights reserved. Introduction sequences and variations in the iron core structure. Bacteria Ferritin superfamily biomolecules perform the functions of iron storage and metabolism in various organisms that range from mammals to bacteria. This protein consists of a nanosized ferric hydrous oxide core (ca. 8 nm) inside a multisubunit protein shell. An average composition of the iron core was proposed as ferrihydrite with the approximate formula (FeOOH)8 (FeO OPO3H2) or (5Fe2O3 9H2O) including up to about 4500 iron atoms in mammals and a smaller amount in bacteria [1–8]. It is well known that ferritin molecules from different organisms have different amino acid possess three types of iron-depositing ferritin-like proteins, the archetypical ferritin (bacterial ferritin) similar to that usually found in eukaryotes, bacterioferritin which contains hemes, and the DNA-binding dodecameric ferritin. Bacterial ferritin consists of a 24-subunit shell surrounding a cavity of about 7.9 nm which can hold about 2500 iron atoms. Bacterioferritin consists of 24 protein subunits, which are similar but smaller than mammalian and bacterial ferritin subunits, associated with 12 hemes. Inside the protein shell, there is a cavity of about 6 nm which can hold about 1800 iron atoms. The dodecameric ferritin consists of a 12-subunit protein shell surrounding a cavity of about 4.5 nm which can hold about 500 iron atoms. Although the iron core structure in ferritins has been studied for a long time using various structural techniques (see, for instance, [9–14]), these studies suggested different models for its structure ranging from monocrystalline to polycrystalline and to polyphasic with different degrees of crystallinity. 182 I.V. Alenkina et al. / Journal of Molecular Structure 1073 (2014) 181–186 Mössbauer spectroscopy is a powerful technique to study ironcontaining biological molecules including ferritin (for review, see e.g. [15–20]). Mössbauer spectra of various ferritins usually demonstrated a superparamagnetic doublet shape down to low temperatures (below 40 K). Mössbauer parameters obtained for superparamagnetic bacterial ferritins and bacterioferritins at low temperatures in [21–27] were similar to those obtained for mammalian ferritins within the instrumental errors. Superparamagnetic Mössbauer spectra of various ferritins were fitted in three ways: (i) using a model-independent fit with a distribution of quadrupole splitting, (ii) using a one quadrupole doublet fit, and (iii) using a superposition of two or more quadrupole doublets. The fit using one quadrupole doublet should be considered as a rough approximation within the homogeneous iron core model, while the fit using more than one quadrupole doublet may be considered as the fit within the heterogeneous iron core model. Mössbauer spectroscopy with a high velocity resolution, demonstrating substantially smaller instrumental errors than in the case of conventional spectrometers, was recently used to study the human liver ferritin and ferritin-like iron in chicken and human tissues. It showed the possibility to distinguish Mössbauer hyperfine parameters for the iron cores from different sources within the homogeneous iron core model, as well as to fit Mössbauer spectra much better within the heterogeneous iron core model [28–33]. Following this approach, we commenced a study of the ferritin-like iron in bacteria. In this work, we chose a rhizobacterium of the genus Azospirillum (for which, to the best of our knowledge, no studies on ferritin-like iron-containing components have so far been reported); in particular, the widely studied species Azospirillum brasilense (strain Sp245), which can form associations with roots of various higher plants, promoting their growth and development via phytohormone excretion, N2 fixation, and other mechanisms [34]. We present here the results of a comparative study of A. brasilense (strain Sp245) biomass samples prepared in two different conditions and human liver ferritin using Mössbauer spectroscopy with a high velocity resolution. Fig. 1. Transmission electron microscopy image of a cell of the rhizobacterium Azospirillum brasilense (strain Sp245). Materials and methods The bacterium A. brasilense Sp245 (from The Collection of Rhizosphere Microorganisms, [WDCM 1021], Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, Saratov) was cultivated at 31 C for 18 h under aeration on a rotary shaker (180 rpm) in a standard phosphate–malate medium with 0.5 g l 1 NH4Cl as a source of bound nitrogen and 0.070 mM Fe–NTA complex (4.0 mg l 1 Fe) as a sole source of iron. Then the cells were separated from the culture medium by centrifugation (2370g, 30 min) and washed 3 times with sterile saline solution (aqueous 0.85% NaCl). The resulting cell suspension (after the final centrifugation) was divided into two parts. One part was immediately rapidly frozen in liquid nitrogen and then lyophilized (sample 1). The other part was stored (in the form of the wet nutrient-free dense suspension, closed to prevent evaporation and drying) in the plastic sample holder of the Mössbauer spectrometer (see below) at room temperature for 3 days, then rapidly frozen in liquid nitrogen and lyophilized (sample 2). These two different preparation conditions (i.e., cultivation up to the end of the logarithmic growth phase (for 18 h) for sample 1, and the same with an additional storage for 3 days in a wet dense nutrient-free saline suspension for sample 2) were applied, as the additional stresses of dense culture and starvation (for sample 2) could be expected to induce some metabolic transformations in the living cells, possibly involving the iron-containing components. A lyophilized normal human liver ferritin containing about 20 wt.% of bound iron (with natural abundance of Fe) was obtained from the Russian State Medical University, Moscow, Russian Federation (the process of ferritin preparation was described elsewhere [35]). For the present study, lyophilized bacterial biomass and human liver ferritin were used as powders with sample weights of 50 mg (bacterial sample 1), 70 mg (bacterial sample 2) and 100 mg (human liver ferritin). These powders were placed in Plexiglas sample holders with a diameter of 20 mm and a height of 5 mm and pressed with a Plexiglas cover to exclude vibrations of particles. The thicknesses of these samples were about 14–20 lg Fe/cm for the bacterial samples and about 5–6 mg Fe/cm for ferritin. Transmission electron microscopy (TEM) images of A. brasilense (strain Sp245) were obtained at the Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS (Saratov), using a Libra 120 electron microscope (Carl Zeiss, Germany) at 80 kV. The washed cells were re-suspended in the saline solution up to the initial volume and placed onto nickel grids coated with formvar (1% formvar in dichloroethane). Mössbauer spectra with a high velocity resolution were measured at the Ural Federal University (Ekaterinburg) using an automated precision Mössbauer spectrometric system built on the base of the SM-2201 spectrometer with a saw-tooth shaped velocity reference signal formed by a digital-analog convertor using quantification with 4096 steps. Details and characteristics of this spectrometer and the system are given elsewhere [36–38]. A Co(Rh) source of about 1.8 10 Bq (Ritverc GmbH, St. Petersburg, Russia) was used at room temperature. The standard absorber of sodium nitroprusside with a thickness of 5 mg Fe/cm was used for calibration of the velocity scale. The line width of this spectrum was 0.229 ± 0.003 mm/s with the purely Lorentzian line shape. The Mössbauer spectra of bacterial samples were recorded at 295 K and converted to 2048 channels by consequent summation of two neighboring channels to reach statistics of 5.2 10 counts per channel for sample 1 and 6.4 10 counts per channel for sample 2. The signal-to-noise ratio for the obtained spectra was 27 for sample 1 and 21 for sample 2. The Mössbauer spectrum of human liver ferritin was measured at 295 K in 4096 channels with statistics of 2.7 10 counts per channel and a signal-to-noise ratio of 95. All spectra were computer fitted with the least squares procedure using the UNIVEM-MS program with the Lorentzian line shape. Spectral parameters such as the isomer shift, d, quadrupole splitting, DEQ, line width, C, relative subspectrum area, S, and statistical criterion, v, were evaluated. The criteria for choosing the best fits were differential spectrum, v and the physical meaning of the spectral parameters. The instrumental (systematic) error