Raman difference spectroscopic studies of the myosin S1.MgADP.vanadate complex.

The Raman spectra of the nonbridging V--O bonds in the myosin S1.MgADP.Vi complex, often believed to be a transition-state analogue for the phosphotransfer reaction catalyzed by myosin, and in a vanadate solution model compound have been obtained using Raman difference spectroscopic techniques. A symmetric/asymmetric pair of modes at 870 cm-1 is found for vanadate in solution while three bands are found in the myosin S1.MgADP.Vi complex at 870, 844, and 829 cm-1. Using empirical relationships that relate bond order/bond lengths to stretch frequencies, the bond order and bond length of the three nonbridging V--O bonds of vanadate in solution were determined to be 1.43 vu (+/-0.04 vu) and 1.669 A (+/-0.004 A), respectively. The average bond order and bond length of the nonbridging V--O bonds in the S1.MgADP.Vi complex were determined to be 1.38 vu and 1.683 A. A normal-mode analysis suggests that the VO32- moiety approaches a planar conformation in the enzymic complex. Ab initio calculations show that a water molecule at the S1 ATPase binding site, in line with the apical O-V bond in the ADP-Vi moiety and believed to be the attacking nucleophile in the phosphotransfer reaction, can account well for the changes in frequencies of vanadate when it binds to the protein by forming a moderately strong V-O(H2) bond. Hence, an important role determining the ATPase activity at the active site of myosin appears to be a strategic positioning of this in-line water molecule. Assuming that the distortions that vanadate undergoes upon forming the S1.MgADP.Vi complex are analogous to the changes of the gamma-phosphate of ATP in the transition state of the myosin-catalyzed hydrolysis, our results suggest that this reaction proceeds close to a concerted (SN2-like) process.