Iron metabolism of Escherichia coli studied by Mössbauer spectroscopy and biochemical methods

Abstract

To date it has barely been recognized that the nature of about 75% of the Escherichiu coli iron pool is unknown. Here we report the isolation of two iron species representing major components of iron metabolism in various growth states of E. coli. In vivo Mossbauer spectroscopy was applied to obtain information on the intracellular distribution pattern of iron in E. coli K12 W3110. Only two types of iron could be detected in the cell spectra: hexacoordinated Fe2+ and Fe3 + high-spin complexes. Other iron-requiring compounds are at least one order of magnitude less abundant in E. coli. The Mossbauer parameters of these complexcs fit neither cytochromes nor iron-sulfur proteins nor ferric holo-bacterioferritin. They are sensitive to metabolic changes and inhibitors. The ratio of Fe/subunit, Fe2 '/Fe3 + interconversion, chromatographic and electrophoretic data exclude bacterioferritin as the main iron metabolite in E. coli. Bacterioferritin can be observed only at very high ferric ion concentrations in the medium. The 55Fe fluorograms of both cytoplasmic and membrane fractions exhibit two exclusive bands with apparent molecular masses of 17 and 15 kDa, respectively. The two bands comprised 70% ofthe applied radioactivity. In gel filtration the main iron peak elutes at 155 kDa yielding two bands with apparent molecular masses of 17 and 15 kDa on SDS/PAGE. We therefore conclude that the iron species form a protein with an apparent molecular mass of 155 kDa containing 37-kDa and 15-kDa subunits. The iron content of the protein is 44 pg Fe/mg protein which corresponds to approximately 13 iron ions/subunit. No iron protein cxhibiting the observed features has been described so far. Additional Mossbauer experiments suggest that these novel iron proteins are not restricted to E. coli but that similar components are detectable in several bacterial and fungal systems, thus pointing to a general occurrcnce.Knowledge of the intracellular iron metabolism of both higher and microbial organisms is fragmentary. For Escherichiu coli, the following data are documented (values given per gram cells): total iron content 59-168 pg, nonheme iron in membranes 3.8 -15 pg, heme iron 0.52-0.56 pg [I, 21. Thus, the state of 75% of the E. coli iron pool remains obscure. The quantitative distribution of the metal within the various non-respiratory enzymes and bacterioferritin has not yet been determined. This paper provides evidence that the niain fraction of intracellular iron is associatud with novel components of iron metabolism in E. coli.Prcviously we have shown that in viiw Mossbauer spectroscopy furnishes detailed information on the oxidation state and to some extent on the intracellular distribution pattern of iron in various microbial systems [3 -61. In the present study this technique has been utilized to gain novel insights into the bacterial iron metabolism of E. coli K12 wild-type strain W313 0. In order to achieve specific and controlled uptake of "Fe, we have exploited one feature which E. coli has in common with many microorgan...

Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae.

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