Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) were used to study the motional dynamics and segmental flexibility of cardiac myosin in the presence of free radical generating system. Cardiac myosin isolated from calf heart muscle were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules attached to cardiac myosin decreased with time following a single exponential curve. MgADP and MgADP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. DSC measurements resulted in two main transitions at 49.4 degrees C and 54.1 degrees C, respectively. Addition of MgADP produced a decrease of 49.4 degrees C transition, whereas a shift towards higher temperature was detected at 54.1 degrees C transition. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. The total enthalpy of the UV irradiated myosin decreased significantly in different intermediate states of the ATP cycle.