We use infrared spectroscopy to demonstrate the critical role that trace O-2 plays in determining the products formed when a [2Fe2S] cluster protein reacts with nitric oxide (NO). The observed importance of O-2 may have physiological relevance, as many pathogens sense NO using iron-sulfur proteins and will be exposed to NO in an aerobic environment during a mammalian immune response. We show that the [2Fe2S]-containing spinach ferredoxin I undergoes reaction with NO at pH 6.0, with the proportion of protein-bound Roussin's Red Ester compared to the dinitrosyl iron complex product favored by trace O-2. Roussin's Red Ester is also favored on nitrosylation in the presence of the thiolate scavenging reagent, iodoacetamide, suggesting that the role of O-2 is in oxidative sequestration of cysteine thiolates. Infrared spectroscopy has been overlooked as a tool for studying iron-sulfur protein nitrosylation despite the fact that there exists a wealth of infrared spectroscopic data on small-molecule nitrosyl clusters which serve as models for the identification of protein-bound nitrosyl clusters.