Photoinactivation of the detoxifying enzyme nitrophenol reductase from Rhodobacter capsulatus

Abstract

The phototrophic bacterium Rhodobacter capsulatus E1F1 detoxifies 2,4-dinitrophenol by inducing an NAD(P)H-dependent iron flavoprotein that reduces this compound to the less toxic end product 2-amino-4-nitrophenol. This nitrophenol reductase was stable in crude extracts containing carotenes, but it became rapidly inactivated when purified protein was exposed to intense white light or moderate blue light intensities, especially in the presence of exogenous flavins. Red light irradiation had no effect on nitrophenol reductase activity. Photoinactivation of the enzyme was irreversible and increased under anoxic conditions. This photoinactivation was prevented by reductants such as NAD(P)H and EDTA and by the excited flavin quencher iodide. Addition of superoxide dismutase, catalase, tryptophan or histidine did not affect photoinactivation of nitrophenol reductase, thus excluding these reactive dioxygen species as the inactivating agent. Substantial protection by 2,4-dinitrophenol also took place when the enzyme was irradiated at a wavelength coinciding with one of the absorption peaks of this compound (365nm). These results suggest that the lability of nitrophenol reductase was due to the absorption of blue light by the flavin prosthetic group, thus producing an excited flavin that might irreversibly oxidize some functional group(s) necessary for enzyme catalysis. Nitrophenol reductase may be preserved in vivo from blue light photoinactivation by the high content of carotenes and excess of reducing equivalents in phototrophic growing cells.