Superoxide promotes hydroxyl-radical formation and consequent DNA
 damage in cells of all types. The long-standing hypothesis that it
 primarily does so by delivering electrons to adventitious iron on DNA
 was refuted by recent studies in Escherichia coli.
 Alternative proposals have suggested that superoxide may accelerate
 oxidative DNA damage by leaching iron from storage proteins or enzymic
 [4Fe-4S] clusters. The released iron might then deposit on the
 surface of the DNA, where it could catalyze the formation of DNA
 oxidants using other electron donors. The latter model is affirmed by
 the experiments described here. Whole-cell electron paramagnetic
 resonance demonstrated that the level of loose iron in
 superoxide-stressed cells greatly exceeds that of unstressed cells.
 Bacterial iron storage proteins were not the major source for free
 iron, since superoxide also increased iron levels in mutants lacking
 these iron storage proteins. However, overproduction of an enzyme
 containing a labile [4Fe-4S] cluster dramatically increased the free
 iron content of cells when they were growing in air. The rates of
 spontaneous mutagenesis and DNA damage from exogenous
 H2O2 increased commensurately. It is striking
 that both growth defects and DNA damage caused by superoxide ensue from
 its ability to damage a subset of iron–sulfur clusters.

https://www.pnas.org/content/pnas/93/24/13635.full.pdf

Superoxide accelerates DNA damage by elevating free-iron levels

The Fur (ferric uptake regulation) protein is a negative regulator of the aerobactin operon and of several other siderophore-mediated, high-affinity iron transport systems in Escherichia coli. The purified Fur protein and a plasmid containing a lacZ fusion to the aerobactin operon were used in conjunction with an in vitro coupled transcription/translation system to demonstrate that the Fur protein requires Fe(II) or certain other divalent metals as a cofactor to negatively regulate expression of the aerobactin operon. In a second set of experiments, using a restriction site protection assay, Fur was shown to bind to and block the aerobactin promoter in a metal-dependent fashion. It is concluded that Fur acts as a classical negative repressor that, under in vivo conditions, uses ionic Fe(II) as a corepressor. Our results support the hypothesis [Williams, R.J.P. (1982) FEBS Lett. 140, 3-10] that prokaryotic cells may contain a standing pool of free or loosely bound Fe(II) that is capable of acting in a regulatory capacity.

Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli.

Ferritin and superoxide-dependent lipid peroxidation.

A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from Azotobacter vinelandii and Clostridium pasteurianum is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from A. vinelandii contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C(2)H(4) formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in A. vinelandii mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa, and Rhodospirillum rubrum activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type A. vinelandii derepressed for nitrogenase synthesis in tungsten-containing medium and K. pneumoniae mutant strain UN109 are also activated in vitro by FeMoCo. The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in N-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.

https://www.pnas.org/content/pnas/74/8/3249.full.pdf

Isolation of an iron-molybdenum cofactor from nitrogenase

Tight-binding inhibitors—I: Kinetic behavior☆

Studies on Milk Xanthine Oxidase: Some spectral and kinetic properties

Publisher Summary This chapter describes the methods for the determination of nonheme iron, total iron, and copper. Nonheme iron may be extracted quantitatively from most materials with trichloroacetic acid, example NADH dehydrogenase, succinie dehydrogenase, and the liberated iron. For the determination of total iron in biological samples, the material for analysis is most commonly subjected to an acid digestion prior to carrying out the color reaction. A number of different methods for digesting the sample and developing the color are described (methods A, B, and C). A method is also described for the determination of total iron using an extraction procedure in place of the acid digestion (method D). There are a number of reagents, which form more or less specific colored complexes with copper, so that interference can usually be overcome by selection from the methods of determination available. This is especially so for biological samples which are generally free from interfering elements, and preliminary separations are seldom required. As for iron determinations, the color reaction with copper is usually preceded by digestion of the sample (methods A and B). An extraction method has also been described, and has been used to determine the valency state of the copper in the sample.

Philip E. Brumby

Papers

Studies on Milk Xanthine Oxidase: Some spectral and kinetic properties

[73] Determination of nonheme iron, total iron, and copper

The content and possible catalytic significance of labile sulfide in some metalloflavoproteins.

The reversible conversion of lipoyl dehydrogenase to an artifactual enzyme by oxidation of sulfhydryl groups.