The Use Of Glassy Matrices To Identify Intermediates In The Nitric Oxide Dioxygenase Reaction Of Hemoglobins And Myoglobins

Abstract

The nitric oxide dioxygenase (NOD) reaction of nitric oxide with ferrous oxy derivatives of Hb and Mbs is important from many physiological standpoints. Several mechanisms have been proposed; however, the experimental difficulties in probing this reaction in solution at physiological pH and ambient temperatures have precluded an unambiguous determination of the sequence and nature of intermediates. We have developed a method of following the progression of this reaction in glassy matrices that allows for the trapping and probing of key intermediates. The technique is based on incorporation of O2 derivatives of Hb and Mb in a thin glassy matrix (derived from trehalose) that lines the inner wall of an optical quality tube. After purging the sample with dry nitrogen to remove the unbound excess oxygen, the tube is filled with NO. Absorption spectroscopy is used to follow the spectral progression initiated as the NO slowly accesses the heme bound oxygen. The spectra reveal an intermediate that resembles the spectrum attributed to the bound peroxynitrite intermediate. The final product under these condition is a species with a spectrum that is identical to that which is generated when met Hb(Mb) is incorporated into a glass in the presence of an excess of nitrate. The spectrum attributed to the nitrate product only appears when the glass is dry. The results are consistent with water being an effective competitor for the ferric heme site in the presence of nitrate.