Abstract
Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.
Highlights
Cytochromes cd1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer
Activity is associated with histidine/methionine coordination at heme c, and it is believed that Pseudomonas stutzeri (Ps). pantotrophus cytochrome cd1 is unreactive toward substrate without reductive activation
Crystallography further showed that reduction of both hemes to the FeII state triggers a conformational change, which results in the replacement of the heme c ligand His17 with Met106 and the dissociation of Tyr25 from heme d1 (6), creating a vacant coordination site at which substrate can bind
Summary
Purification of cd1—Samples of cd from Pp and Pseudomonas stutzeri (Ps) strain ZoBell (ATCC 14405) were purified according to published methods (10, 31). Samples of the Pp cd1*-X were prepared according to reported methods (11, 29): oxidized Pp cd was reduced in an anaerobic hood (Faircrest Engineering; operating at Ͻ1 ppm O2 in an N2 atmosphere) using aliquots of buffered solutions of sodium dithionite; excess reductant was removed by chromatography on a PD-10 desalting column; the enzyme was reoxidized using aliquots of buffered hydroxylamine solution. Aliquots of buffered potassium nitrite solutions, freshly prepared, were added within 10 –15 s of this reoxidation. Assays of nitrite reductase activity were performed following published methods (13) using reduced equine cytochrome c as electron donor. Samples for MCD spectroscopy were prepared in deuterium oxide solutions containing 50 mM BTP buffer at pH* ϭ 6.5 to which equivalent volumes of glycerol had been added as glassing agent (33). Samples for MCD spectroscopy were prepared in deuterium oxide solutions containing 50 mM BTP buffer at pH* ϭ 6.5 to which equivalent volumes of glycerol had been added as glassing agent (33). pH* is the apparent pH of the D2O-based solutions measured using a standard glass pH electrode
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