The manganese binding site of chloroplast coupling factor one as revealed by paramagnetically induced nuclear magnetic relaxation.

The Mn(II) binding sites of latent soluble spinach chloroplast coupling factor one (CF$\sb1$) were characterized through studies of the paramagnetic NMR spin-lattice relaxation rates of solvent water protons. First, the affinity of CF$\sb1$ for Mn(II) was quantitated by proton relaxation enhancement (PRE), revealing two to three cooperative high-affinity Mn(II) binding sites and several low-affinity Mn(II) binding sites. Because of the positive cooperativity between high-affinity sites, a PRE analysis for cooperative systems was developed. The enhancement of the high-affinity sites, $\epsilon\sb{\rm b}$, was 9.01 $\pm$ 0.11 and the apparent dissociation constant to site ratio, K/n, was 4.6-5.3 $\mu$M. Next, the environment of the first site which Mn(II) occupied was studied by variable magnetic field strength measurements. Proton relaxation rates at frequencies between 4 and 62 MHz were fitted, using nonlinear least-squares analyses, to the Solomon-Bloembergen-Morgan equations. The analyses gave chemical information about the inner coordination sphere of bound Mn(II), allowing comparisons of the site in the absence and presence of added adenine nucleotides. Proton and deuteron relaxation rates measured at the same frequency provided starting values for several parameters and a unique fit to the magnetic field dependence data. To accommodate both deuteron and proton data, a modified expression for electron spin relaxation including terms for high and low frequency ligand field distortions was introduced. In the absence of added nucleotides, the number, q, and residence time, $\tau\sb{\rm m}$, of exchangeable water molecules within the Mn(II) coordination sphere were one and 330 ns, respectively. Little field dependence in the dipolar correlation time, $\tau\sb{\rm c}$, was found. In the presence of added ADP and ATP, the numbers of exchangeable water molecules were three and two and the residence times 430 and 260 ns, respectively. Strong field dependence in $\tau\sb{\rm c}$ was evident, as reflected in distinct local relaxation rate maxima. The results indicate that in the presence of added adenine nucleotides Mn(II) bound as Mn(II)-nucleotide complexes, while in their absence Mn(II) bound with more protein ligands to a site not occupied by nucleotide. The identity of the sites and the mode of Mn(II) binding to CF$\sb1$ are discussed.Ph.D.BiochemistryBiological SciencesBiophysicsPhysical chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128154/2/8812903.pd