ELECTRON-NUCLEAR COUPLING TO THE PROXIMAL HISTIDINE IN OXY COBALT-SUBSTITUTED DISTAL HISTIDINE MUTANTS OF HUMAN MYOGLOBIN

Electron spin echo envelope modulation (ESEEM) spectroscopy was used t o investigate electron-nuclear coupling to the N-epsilon of the proxim al histidine (F8, His 93) imidazole in oxyCo(II)-substituted distal hi stidine (E7, His 64) mutants (His --> Leu, His --> Val, His --> Gln) a nd recombinant wild-type human myoglobins (Mbs). Nuclear hyperfine and nuclear quadrupole coupling constants decrease in the order: H64L > H 64V greater than or equal to H64G approximate to H64Q > wild-type. The differences in couplings found for the four mutant proteins are corre lated with the differences in polarity of the E7 side chain. On the ba sis of the relative orientation of the nuclear quadrupole and g tensor s, obtained by computer simulation of ESEEM spectra, the Co-O-O bond a ngle of H64Q and H64Q appears to be similar to that of oxyCo sperm wha le Mb (and possibly wild-type human Mb) at room temperature [Hori et a l. (1982) J. Biol. Chem. 257, 3636], while that in H64V and H64L is mo re obtuse. ESEEM measurements in D2O demonstrate the presence of a hyd rogen bond between the distal histidine and bound O-2 in the wild-type protein, as was found in oxyCo sperm whale and horse Mbs [Lee et al. (1992) Biochemistry 31, 7274]. This hydrogen bond leads to a reduction in the N epsilon coupling in the wild-type protein as compared to tha t in the E7 mutants. No hyperfine-coupled deuterons were found in any of the mutants, and therefore, the proposed hydrogen bond between boun d O-2 and the distal glutamine in H64Q [Ikeda-Saito et al. (1991) J. B iol. Chem. 266, 23641] could not be substantiated.