Glass matrix-facilitated thermal reduction: A tool for probing reactions of met hemoglobin with nitrite and nitric oxide

Isolating elemental steps that comprise a protein reaction in solution is a difficult process. In this study, the use of sugar-derived glass matrices is evaluated as a biophysical tool to help dissect out elemental steps and isolate intermediates. Two features of the glass are utilized in this endeavor: (i) the capacity of trehalose glass matrices to support thermal reduction over macroscopic distances; and (ii) the ability of glass matrices to significantly damp large amplitude protein dynamics. The focus of the study is on the reaction of nitric oxide (NO) with a nitrite ion coordinated to the heme iron of hemoglobin (Hb). The thermal reduction property of the glass is used to generate NO from nitrite within the glass, and the damping of protein dynamics is used to control entry of NO into the distal heme pocket of Hb, where it can either interact with bound nitrite or bind to the heme iron. The results not only relate to earlier controversial studies addressing the reactions of Hb with NO and nitrite but also raise the prospect that these properties of sugar-derived glassy matrices can be exploited as a new biophysical tool to modulate and probe reactions of NO with hemeproteins as well as a wide range of other metalloproteins.