F-19 AND H-1,H-2 ENDOR STUDY OF DISTAL-POCKET N(EPSILON)-H-CENTER-DOT-CENTER-DOT-CENTER-DOT-F HYDROGEN-BONDING IN FLUOROMETMYOGLOBIN

F-19 and H-1,H-2 continuous-wave and pulsed electron nuclear double re sonance (ENDOR) measurements of fluorometmyoglobin (MbF) frozen soluti ons are shown to provide probes of subtle structural features at the h eme that are not directly accessible through X-ray methods. Although t he EPR spectrum of MbF is pH invariant, the F-19 data show that MbF ex ists in low- and high-pH forms that are related by a one-proton equili brium with pK(a) 7.6(1). The H-1,H-2 ENDOR shows that F- is hydrogen-b onded to the N(epsilon) H of distal histidine (E7) at all pH values be tween 5.5 and 11. The pH-dependent interconversion is assigned to depr otonation at N(delta) of the charged (imidazolium) form of His(E7). Th e data further gives metrical parameters for the H-bond, showing it to be relatively short with r(F ... H) 1.57(5) Angstrom. Complete determ ination of the F-19 hyperfine tenser discloses a slight tilt of Fe-F- (non-coaxiality of g and hyperfine tensors). The tilt is associated wi th the H-bond: it is 5.0(5)degrees in the low-pH form and 3.5(5)degree s in the high. The F-19 tenser components change slightly upon deproto nation: low pH, A parallel to = +127.0(4) MHz, A perpendicular to = +6 4.0(4) MHz; high pH, A parallel to = +130.0(4) MHz. A perpendicular to = +67.5(4) MHz. Surprisingly, there is a deuterium isotope effect on the F-19 coupling in the low-pH form. The signs of the hyperfine compo nents are derived from the pseudonuclear Zeeman effect in the F-19 END OR. This effect also gives to high precision the axial zero-field spli tting parameter, D, whose value is determined by the energies of excit ed states. D also is pH dependent: low-pH form, D = 6.1(1) cm(-1); hig h-pH form, D = 5.2(1) cm(-1).