Characterization of interactions of nitric oxide with human hemoglobin A by infrared spectroscopy

Infrared spectra permit direct measurements of cysteine thiols as well as nitric oxide bound to heme iron in human hemoglobin A nitrosyl. A single symmetric N-O stretch band of nitric oxide bound to Fe2+ is detected amid strong water and protein bands in the Hb14N16O minus Hb15N16O difference spectrum. Nitric oxide accepts electron density from metal in bent-end-on FeI2+)-14N-16O (nu NO = 1616.5 cm-1) and donates electron density to metal in linear Fe(3+)-14N-16O (nu NO = 1925 cm-1). S-H stretch bands reveal that changes in protein conformation occur at alpha-104, beta-93, and beta-112 cysteines upon conversion of deoxyHb to HbNO but that no reactions of thiols with NO occur. Furthermore, no infrared band for S-nitrosothiol is detected. Changes in amide I spectra reflect NO binding induced changes in protein secondary structure.