Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin

We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two‐headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at ∼5 pN of retroactive force. Using microtubule‐activated release of the fluorescent analogue 2′‐(3′)‐O‐(N‐methylanthraniloyl) adenosine 5′‐diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide ‘chase’. mantADP was released from the second head at 4 s−1 by an ADP chase, 5 s−1 by 5′‐adenylylimidodiphosphate (AMPPNP), 27 s−1 by ATPγS and 60 s−1 by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15‐fold, and leading head binding then accelerates trailing head unbinding at least 6‐fold.