Abstract
Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.
Highlights
Depending on the concentration, nitric oxide (NO) has different roles in living systems [1]
One of these mechanisms involves a family of cytoplasmic enzymes named flavodiiron proteins (FDPs), which are capable of protecting cells by reducing O2 and/or NO to water and nitrous oxide, respectively: O2 + 4e− + 4H+ → 2 H2 O
With the large number of FDP amino acid sequences available, it is known that this motif is less conserved and shorter
Summary
Nitric oxide (NO) has different roles in living systems [1]. One of the defense mechanisms used by the mammalian innate immune system to fight pathogens is based on the production of NO through the inducible nitric oxide synthases in phagocytic cells, namely macrophages [2]. To tackle the effects of NO and O2 exposure, organisms have acquired throughout evolution enzymatic mechanisms capable of removing these potentially harmful molecules. One of these mechanisms involves a family of cytoplasmic enzymes named flavodiiron proteins (FDPs), which are capable of protecting cells by reducing O2 and/or NO to water and nitrous oxide, respectively (for recent views, see [6,7]): O2 + 4e− + 4H+ → 2 H2 O (1)
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