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.

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The branching of the electron flow to individual teminal acceptors NO3−, NO2− and N2O was investigated in denitrifying cells of Paracoccus denitrificans. It was found that the extent of electron flow to nitrate depends on the activity of electron flow through the terminal part of the respiratory chain comprising cytochrome c. NO2− and N2O either endogenously formed or exogenously added decreased the rate of nitrate reduction by oxidizing the respiratory chain via nitrite reductase and nitrous oxide reductase. The inhibitory effect of both intermediates of denitrification was released by antimycin. The rate of the first reaction of the sequence NO3−→ NO2−→ N2O → N2 is in the type of cells used under feedback redox control exerted by concentration of intermediates. This regulation can be designated as ‘inhibition by product via respiratory chain’. The reaction rate of nitrate denitrification could also be influenced by changing the portion of electron flow from substrate to cytochrome c by artificial electron acceptors and donors. By varying the concentration of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine in the presence of ascorbate, the desired redox state of cytochrome c and consequently various rates of nitrate reductase activity could be established. The basis of the enhancing effect of dithionite on the rate of nitrate reduction could also be explained by its functioning as an electron donor for the terminal part of respiratory chain. The quantitative evalution of the extent of electron flow revealed that both branches of the respiratory chain (to nitrate reductase and to cytochrome c) compete for a constant limited flow of redox equivalents supplied from dehydrogenases. A strong evidence exists that the same mechanism is implied in the inhibition of nitrate reduction brought about by oxygen.

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.

A periplasmic protein able to transfer electrons from cytoplasmic membrane to the periplasmic nitrite reductase (cytochrome cd 1 ) has been purified from the anoxically grown cytochrome c 550 mutant strain Pd2121 and shown to be pseudoazurin by several independent criteria (molecular mass, copper content, visible spectrum, N-terminal amino acid sequence). Under our assay conditions, the half-saturation of electron transport occurred at about 10 W WM pseudoazurin; the reaction was retarded by increasing ionic strength.z 1999 Federation of European Biochemical Societies.