Myosin Nucleotide Pocket Thermodynamics Measured by Epr Reveal How Energy Partitioning Relates Speed to Efficiency

Abstract

We have used spin labeled ADP to investigate the dynamics of the nucleotide-binding pocket in myosins. In actomyosin•ADP the nucleotide-binding pocket is in an equilibrium between closed and open conformations, with the open conformation favored in slow myosins. In rabbit slow skeletal muscle fibers, the ∈″G for the closed to open equilibrium is −3.9 kJ mol−1. We found similar values for pig ventricle myosin, chicken gizzard smooth muscle myosin, and chicken myosin V. For faster myosins, the equilibrium shifts to favor a closed conformation, rising to −2.7 kJ mol−1 for Dictyostelium discoideum myosin II, −1.9 kJ mol−1 for pig atrial myosin, −1.1 kJ mol−1 for rabbit fast skeletal muscle fibers, and +2.9 kJ mol−1 for Drosophila fight muscle fibers. We believe this represents a destabilization of the open actomyosin•ADP state in the faster myosins, driving ADP release. van't Hoff analysis of the temperature dependence of this equilibrium reveals that the closed to open conformation has a significant positive enthalpy and entropy, with ∈″H and T∈″S of 40-50 kJ mol−1 for slow myosins. Both components are reduced in this equilibrium for faster myosins, decreasing to ∈″H = 17.7 kJ mol−1 and T∈″S at 25°C = 18.8 kJ mol−1 for rabbit fast skeletal fibers, and ∈″H = 10.4 kJ mol−1 and T∈″S at 25°C = 7.5 kJ mol−1. Our model is that the open actomyosin•ADP state represents a partitioning point between the free energy released during the myosin catalytic cycle. Because of this partitioning, fast myosins destabilize the actomyosin•ADP state, reducing the energy available to do work up until that point, but leaving more free energy in reserve to drive ADP release. This gives a mechanism for the correlation between increased speed and reduced efficiency in muscle.