* To whom correspondence should be addressed.
Received January 24, 2014; Revision received June 5, 2014
The pH dependence of proteins is related to the thermodynamic stability and electrostatic interactions in the native state of a protein. Here we report the pH-induced conformational transition of the heme protein leghemoglobin (Lb) isolated from root nodules of the leguminous plant Arachis hypogea. Unlike the other heme proteins myoglobin, hemoglobin, and cytochrome c, the structural characteristics and interactions of Lb is almost unknown, though its functional importance is already established since it binds oxygen to maintain the environment for N2 fixation. We investigated pH-induced unfolding of this protein and identified a number of conformational isomers using multiple fluorescence observables as a function of pH titration. We have characterized the acid- and base-induced conformational transitions among the structural states over the pH range 2-11. Depending on the solution conditions, Lb can exist in one of three phases: pH 2, 3, 4; pH 5, 6, 7; pH 8, 9, 10. The secondary structure as revealed by CD spectroscopy indicated the maximum percentage of α-helix to be present at pH 7, where the structure of Lb is also most rigid according to fluorescence anisotropy experiments. The fluorescence lifetime of tryptophan was observed to be maximum at pH 10 and minimum at pH 6, suggesting unfolding transitions of Lb. Thus, alteration of the microenvironment of the globin moiety during pH transition ultimately leads to the conformational change of this monomeric protein Lb.
KEY WORDS: leghemoglobin, pH titration, protein conformation, unfolding, mean residual ellipticity, excited state lifetime, microenvironment