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.