Homodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structuralanalysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structuralparameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI.
(a) Kinetic model for T72V HbI. Time constants for T72V, wild type, and F97Y are represented in black, red, and blue, respectively. The red (with “CO”) and white symbols represent ligated and photolyzed subunits, respectively. To indicate the change in tertiary structure with the progress of structural transition, we represented the subunits of each intermediate with symbols of different shapes. To indicate the change in quaternary structure in the transition from I2 to I3, we described the two subunits of I3 rotating with respect to each other. Two red octagons represent a ligated form of I1, which is formed by geminate recombination of CO with I2 and is structurally indistinguishable from the photolyzed forms of I1. (b) Species-associated difference scattering curves for the three intermediates of T72V (black), wild-type (red), and F97Y (blue) HbI.