Laser flash photochemical oxidation to locate heme binding and conformational changes in myoglobin

Fast photochemical oxidation of proteins (FPOP), a means of protein footprinting, uses OH radicals to oxidize the solvent-exposed residues of proteins on a short time scale (∼1 μs). A 248 nm pulsed laser beam dissociates H2O2 (15 mM) into hydroxyl radicals. The radicals react with exposed and reactive amino acid residues (e.g., C, M, W, Y, F, H, L, I) in competition with quenching with a glutamine scavenger present in solution. We report here the use of FPOP to confirm that the F-helix is conformationally constrained in the holo form of myoglobin, whereas it is conformationally free in the apo form. The interpretation finds support in the differences in oxidation of various residues in the apo versus holo forms. The differential reactivity of leucine 137 suggests that it is part of a hinge region on the H-helix, enabling the binding pocket to close in the apo form. This is our second study that offers support that FPOP is capable of elucidating the conformational dynamics of proteins by obtaining information that may not be accessible by other methods.