The multiple pathways of the actin-myosin cycle of energy transduction and the release of orthophosphate in muscle

Marco Caremani¹Irene Pertici²Ilaria Morotti¹Pasquale Bianco¹Massimo Reconditi³Gabriella Piazzesi⁴Vincenzo Lombardi^4*Marco Linari^1*

• ¹PhysioLab, Department of Biology, University of Florence, Florence, Italy
• ²PhysioLab, University of Florence, University of Florence, Florence, Italy
• ³PhysioLab, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
• ⁴PhysioLab, University of Florence, Florence, Italy

In the striated muscle the molecular motor myosin II works in two bipolar arrays in each thick filament, converting chemical energy into steady force and shortening by cyclic ATP–driven interactions with the nearby actin filaments. The fundamental steps in the energy transduction are the working stroke, an interdomain tilting of the lever arm about the actin-attached catalytic domain, generating up to ∿5 pN force or ∿10 nm filament sliding, and the release of the ATP hydrolysis product orthophosphate (Pi) from the nucleotide-binding site, associated with large free energy release. The two events are not simultaneous, as first demonstrated by the force response to stepwise change in [Pi] (the Pi transient), showing a saturation kinetics characteristic of a two-step reaction. However, while high resolution crystal structures of the myosin motor suggest that Pi release precedes the working stroke, in vitro functional studies indicate that it follows the working stroke. High resolution sarcomere level mechanics applied to single muscle fibres, allowing myosin motor synchronization by step perturbations in length or load, revealed that the working stroke kinetics is independent of [Pi] and only depends on the load. Moreover, this approach highlights the need for two unconventional pathways of the chemo-mechanical cycle: an early detachment of the force-generating motors and the possibility for attached motors to slip to the next actin monomer farther from the sarcomere centre during shortening. Transient and steady state responses to stepwise changes in load or [Pi] can be fitted with a structurally and biochemically explicit model in which the Pi release step is orthogonal to the progression of the working stroke. Model simulation indicates that the rate of Pi release depends on the motor conformation, which solves longstanding unanswered questions such as the dependence of the Pi transient kinetics on the final level of [Pi] under whatever load and clarifies the issue of the relative timing between the working stroke and Pi release: at high loads release of Pi leads the execution of the working stroke, while at low loads the working stroke state transitions are fast enough to occur with Pi bound to the catalytic site.