Reactive radical molecules, such as nitric oxide (NO) have to be protected in order to avoid side reactions with its surrounding environment. A central question in NO biology involves understanding how this protection occurs and what chemistry is involved in side reactions with oxygen and other environmental compounds. The primary function of the nitrophorins, a family of proteins that aid in feeding of the blood sucking insect Rhodnius prolixus, is to store, transport, and release NO. Previous structural analysis of Rhodnius nitrophorin 4 (rNP4) revealed a substantial NO-induced conformational change in which two poorly ordered loops collapse into the binding pocket, expel water, and pack nonpolar side chains around the heme-ligated NO moiety. We have created a triple alanine mutant (AAA) of the rNP4 (V36A/D129A/L130A) loop region and obtained crystallographic data and kinetic measurements using stopped-flow and UV-visible spectrophotometry. A low pH, NO ligated crystal structure refined to 1.0 angstroms shows the loop network is destroyed and closure is abolished. Aerobic stopped-flow measurements show that NO reacts much faster with the heme iron than with surrounding oxygen in both the wild type and AAA mutant proteins. Aerobic UV-visible measurements show that loop closure leads to a large level of protection of NO from surrounding oxygen. In addition, NO release is accelerated by the addition of glutathione, a naturally occurring thiol compound found in the victim's plasma. Possible mechanisms for this activity will be discussed.