Quantifying the photoinduced release of nitric oxide from N,N'-bis(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine. Effect of reducing agents on the mechanism of the photoinduced reactions

N,N'-Bis(carboxymethyl)-N,N'-dinitroso-1,4-phenylenediamine (1) fragments to release 1 equiv of NO* and the denitrosated radical of 1 (2), when exposed to a approximately 10 ns, 308 nm laser pulse. Species 2 can fragment to give another equivalent of NO* and the doubly denitrosated quinoimine derivative of 1 (3), it can recombine with NO* to give 1 and ring-nitrosated isomers of 1, or in the presence of a reducing agent, 2 can be reduced (to species 4). Photogenerated NO* can be used to probe fast reactions of biochemical interest, making 1 a valuable research tool. This paper focuses on the chemistry of 2, whose reactivity must be well characterized if 1 is to be used to its full potential. [Ru(NH3)6]2+ (RuII) and [Fe(CN)6]4- (FeII) were both shown to reduce 2, with bimolecular rate constants in the diffusion limit. When solutions initially containing 70 microM of RuII, 20 microM myoglobin (Mb) and varying amounts of 1 were irradiated, the only Mb reaction product was nitrosomyoglobin (MbNO). In contrast, in solutions containing only Mb and 1, Mb is converted to both MbNO and oxidized myoglobin (metMb). When FeII was used in place of RuII, Mb was oxidized to metMb, but approximately 100x more slowly than in solutions containing only Mb and 1. This showed that 2 first oxidized FeII to [Fe(CN)6]3- (FeIII), which then oxidized Mb at the slower rate. The ratio metMb/MbNO obtained in the experiments with FeII was 0.6, whereas the ratio predicted from previously known chemistry of 2 was approximately 1 under the experimental conditions. The result is explained if, upon photolysis, 1 first forms a caged encounter complex [2, NO*], which fragments to give 3 and 2 equiv of NO*, without ever releasing free 2 into solution. This hypothesis was further strengthened by analyzing the amount of NO* generated by photolysis of 1 in the absence of added reductant. The original mechanism underestimates the NO* generated, a problem solved by invoking direct release of NO* and 3 from photolysis of 1.