Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron‐limited culture of Paracoccus denitrificans using a combination of anion‐exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox‐active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping‐pong type kinetic pattern with NADH and Fe(III)‐nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N‐terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone‐0 (Q0) or unsubstituted p‐benzoquinone.
In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, b-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of b-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic b-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N 2 O. Additions of nitrate or nitroprusside lowered b-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.
Facultatively anaerobic bacteria are able to adapt to many different growth conditions. Their capability to change their metabolism optimally is often ensured by FNR-like proteins. The FNR protein of Escherichia coli functions as the main regulator during the aerobic-to-anaerobic switch. Low oxygen tensions activate this protein which is expressed constitutively and is inactive under aerobic conditions. The active form is dimeric and contains a [4Fe-4S]2+ cluster. The direct dissociation of the cluster to the [2Fe-2S]2+ cluster by the effect of oxygen leads to destabilization of the FNR dimer and to loss of its activity. The active FNR induces the expression of many anaerobic genes; the set comprises over 100 of controlled genes. Many other bacteria contain one or more FNR analogues. All these proteins form the FNR family of regulatory proteins. Properties of these proteins are very distinct, sometimes even among representatives of different strains of the same bacterial species. FNR-like proteins together with other regulators (e.g., two-component system ArcBA, nitrate-sensing system NarXL, etc.) control a complicated network of modulons that is characteristic for every species or even strain and enables fine tuning of gene expression.
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