An electron spin-echo envelope modulation (ESEEM) study of the Q(A) binding pocket of PS II reaction centers from spinach and Synechocystis

We have used electron spin-echo envelope modulation spectroscopy (ESEEM) to characterize the protein-cofactor interactions present in the Q(A)(-) bind ing pocket of PS II centers isolated from spinach and Synechocystis, We con clude that the ESEEM spectrum of QA- is the result of interactions of the S = 1/2 electron spin of Q(A)(-) With the I = 1 nuclear spins of the peptide nitrogens of two different amino acids. One peptide nitrogen has ESEEM pea ks near 0.7, 2.0, 2.85, and 5.0 MHz with isotropic and dipolar hyperfine co uplings of A(iso) = 2.0 MHz and A(dip) = 0.25 MHz, respectively. On the bas is of these hyperfine couplings we predict the existence of a strong hydrog en bond between Q(A)(-) and the peptide nitrogen with a hydrogen bond dista nce of about 2 Angstrom. We have not identified the amino acid origin of th is peptide nitrogen. By using amino acid specific isotopic labeling in conj unction with site-directed mutagenesis, we demonstrate that the second pept ide nitrogen is that of D2-Ala260, with ESEEM peaks near 0.6 and 1.5 MHz an d an isotropic hyperfine coupling, A(iso) less than 0.2 MHz, This small iso tropic coupling suggests that the D2-Ala260 peptide nitrogen at best forms a weak hydrogen bond with Q(A)(-).