Hydrogen Exchange Studies of Respiratory Proteins: III. STRUCTURAL AND FREE ENERGY CHANGES IN HEMOGLOBIN BY USE OF A DIFFERENCE METHOD

Abstract

Abstract A difference hydrogen exchange method was developed and applied to hemoglobin. The method can be used to pick out, from the total hydrogen exchange curve of a protein, the hydrogen exchange behavior of just those segments of the protein that alter in exchange rate in response to structural change. With the method even quite subtle structure changes can be individually recognized since protein mechanisms act to translate structure changes into greatly amplified hydrogen exchange signals. A fraction of hemoglobin's hydrogen exchange curve containing about 40% of the protein's hydrogen-bonded peptide groups was analyzed. Among these the difference techniques portrayed the exchange behavior of 20 ligand-responsive hydrogens in reduced and liganded hemoglobin and also the exchange of 30 ligand-indifferent hydrogens. The ligand-responsive hydrogens fall into three kinetically distinct groups having 3, 8, and 9 hydrogens on a per subunit basis. All three groups experience much faster exchange in the liganded form of the protein. The exchange character of these groups demonstrates that their exchange, and probably protein hydrogen exchange in general, is controlled by breathing reactions—cooperative, reversible denaturations—of the segments of protein structure holding them. This picture leads immediately to a quantitative relationship connecting energetic structure change, breathing change, and alteration in hydrogen exchange rate. The results, interpreted in this way, show that when ligand is bound to hemoglobin the two larger responsive segments studied are destabilized by close to 2 Cal in structural free energy and the smaller one by perhaps 4 Cal.