Investigations of equilibrium complexes of myoxin subfragment 1 with the manganous ion and adenosine diphosphate using magnetic resonance techniques.

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Electron paramagnetic resonance spectroscopy and water proton relaxation rate (PRR) measurements were used to characterize a complex formed at the myosin subfragment 1 (S1) ATPase site with stoichiometric amounts of Mn(II) and ADP. In the absence of nucleotide, Mn(II) binding at the active site is very weak, although two other classes of sites for Mn(II) on subfragment 1 were identified which are not directly involved in the ATPase reaction. A high affinity Mn(II) site (termed L-site with KL = 3 muM) is associated with a region of the molecule which is susceptible to proteolysis (probably the LC2 light chain subunit) since its stoichiometry depends on the conditions employed for the preparation of subfragment 1 during the papain treatment of myosin. In addition there are a number of weak sites for Mn(II) (termed N-sites) probably associated with anionic groups on the surface of the molecule. In order to study the properties of Mn(II) and ADP bound at the active site by magnetic resonance techniques, subfragment 1 preparations virtually free of the L-site were used, since such an ancillary site competes for the available Mn(II). MnADP binds to subfragment 1 with an apparent dissociation constant, KT, of about 4 muM at 25 degrees. The resultant complex, S1-MnADP, has a low PRR enhancement factor (1.7 at 24.3 MHZ), and its frequency (magnetic field) dependence indicates that this is because there are no readily exchangeable water molecules within the first coordination sphere of Mn(II. Relaxation of the bulk solvent is mediated by protons bound transiently within the outer spheres (4 to 7 A) of the Mn(II). A nitroxide spin label attached to the reactive thiol group of subfragment 1 enhances the solvent PRR, and this property is sensitive to the binding of MgADP to the active site. However, no dipolar spin-spin interaction was detected between the nitroxide group and Mn(II) in the S1-MnADP complex, indicating that the metal ion and thiol group are well separated.