2-DIMENSIONAL NMR CHARACTERIZATION OF THE DEOXYMYOGLOBIN HEME POCKET

Traditionally, assigning the heme protein resonances has relied heavil y on the comparison of spectra arising from protein reconstituted with specifically deuterated hemes and the native form. Such an approach c an identify tentatively the broad, overlapping signals in the Fe(II) h igh-spin heme protein spectra. Although 2D NMR studies have reported a lternative approaches to detect and assign paramagnetic signals, their effectiveness is limited primarily to Fe(III) low-spin systems and st ill depends upon isotopic labeling results to be definitive. For deoxy myoglobin, the reported 2D techniques have not produced any spin corre lation maps. Nevertheless, our study demonstrates that the deoxymyoglo bin spin correlations are indeed detectable and that a complete heme a ssignment, except for the meso protons, is achievable with only 2D NMR and saturation-transfer techniques. The 2D maps improve the spectral resolution dramatically and permit a comprehensive analysis of the deo xymyoglobin signals' temperature dependence, which supports the hypoth esis that the electronic orbital ground state has contributions from b oth (5)E and B-5(2). The results also indicate a structural perturbati on in the vicinity of the 2 vinyl group as the protein undergoes the t ransition from oxy- to deoxymyoglobin state and a significant contribu tion from zero field splitting. Moreover, saturation-transfer experime nts show that NMR can observe directly oxygen binding kinetics.