Non-symbiotic hemoglobin conformational space dependence on the heme coordination using nESI-TIMS-TOF MS.

In this study, for the first time, the conformational space of the rice non-symbiotic hemoglobin type 1 (rHb1) was studied as a function of the starting solution pH using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and molecular dynamics. Comparison of the charge state distribution, apo to holo form ratio, and the collision cross section (Ω) profiles as a function of the solution pH showed higher stability of the rHb1 wild-type (WT) when compared with the H73L mutant at mildly acidic conditions. Comparison of the Ω profiles of the rHb1 WT and H73L holo and apo form showed that only the initial unfolding pathways involved the heme cavity, with and without a heme loss, followed by unfolding pathways not necessarily involving the environment of the heme prosthetic group. Candidate structures for the nine transitions observed in the Ω profiles were proposed using molecular dynamic simulations based on the Ω profiles, UV absorption spectroscopy and circular dichroism data as way to describe a potential unfolding pathway. The described unfolding pathway suggests that the rHb1 unfolding is driven by initial distancing of the A, B, and H helices, while the heme cavity and heme group remains intact, followed by the distancing of the E, F, and G helices and subsequent loss of the α-helical structure leading to a final random coil conformation. [ABSTRACT FROM AUTHOR]