Complexes of phenyldiazene (phenyldiimide) with deoxyhemoglobin and ferroheme

1. 1. Phenyldiazene (C₆H₅N = NH) generated by the method of Huang and Kosower^5-9 through decarboxylation of phenyldiazenecarboxylic acid (C₆H₅N = NCOOH) is shown to form a complex with deoxyhemoglobin. The evidence for complex formation is the greatly reduced reactivity of phenyldiazene (from the complex) towards oxygen. Contrary to the claim of Itano and Robinson (ref. 2), there is no change in the spectrum of deoxyhemoglobin upon combination with phenyldiazene. The binding site is therefore not the iron atom and probably not the heme molecule. It is proposed that a hydrophobic region of hemoglobin complexes with the weakly polar phenyldiazene molecule. 2. 2. Phenyldiazene apparently forms a 2:1 complex with ferroheme. The complex differs in absorption spectrum from ferroheme in all regions of the spectrum. Little or no change in spectrum is noted if freshly prepared complex is exposed to oxygen, reflecting a dramatic decrease in the reactivity of phenyldiazene towards oxygen. 3. 3. Exposure of the phenyldiazene-ferroheme complex to OH^- causes the rapid regeneration of much of the ferroheme, indicating that the normal reactivity of phenyldiazene towards OH^- is retained in the complex. Slow decomposition of the complex near neutral pH is ascribed to reaction with OH^-. 4. 4. The relationship of these phenyldiazene complexes to those of diazene ("diimide", HN = NH) is indicated.