Motions in hemoglobin studied by normal mode analysis and energy minimization: Evidence for the existence of tertiary T-like, quaternary R-like intermediate structures

The normal mode analysis of human hemoglobin showed the presence in the deoxy T-state of one main preferential direction that brings the structure close to the R-state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate T-d1(min), indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to T-d1(min), yielding T-d2(min); then the procedure was repeated, yielding the intermediate structures T-d3(min) and T-d4(min). The structures T-d2(min), T-d3(min) and T-d4(min) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, T-d1(min), is different from the T and R structures, while the second set, T-d2(min), T-d3(min) and T-d4(min), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha(1) beta(1) and alpha(1) beta(2) are R-like, and the alpha and beta heme environments are still T-like.