Mechanistic studies on formation of the dinitrosyl iron complex of the [2Fe-2S] cluster of SoxR protein.

The transcriptional activation of SoxR in Escherichia coli occurs through direct modification of the [2Fe-2S] by nitric oxide (NO) to form a dinitrosyl iron complex (DNIC). Here, we investigated the reactions of NO with [2Fe-2S] clusters of SoxR; the results were compared with those obtained for spinach ferredoxin (Fd). UV-visible absorption and ESR spectra of SoxR upon treatment with an NO donor showed the formation of DNIC of SoxR efficiently, whereas those of Fd exhibited small changes. Upon pulse radiolysis of a deaerated solution of SoxR in the presence of sodium nitrite, a biphasic change in absorption, consisting of a faster phase and a slower phase, was observed. The slower phase fraction was increased with increases in the [NO]/[SoxR] molar ratio, reaching a plateau at ∼2 equivalents of NO. On the basis of these results, we propose that the faster phase corresponds to the reaction of the first NO molecule with [2Fe-2S] of SoxR, followed by the reaction of the second NO molecule. In the reaction of NO with Fd, no slower phase was observed. These results suggest that the reaction of the second equivalent of NO is an important process for the formation of DNIC.

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