Wild-type nitrogenase MoFe protein shows a deep 14N electron spin- echo envelope modulation (ESEEM) arising from a nitrogen nucleus (N1) coupled to the S = 3/2 spin system of the FeMo-cofactor of the MoFe protein. A previous ESEEM study on altered MoFe proteins generated by substitutions at the α-195-histidine positron suggested that α-195-histidine provides a hydrogen bond to the FeMo-cofactor but is not the source of the 14N1 modulation [DeRose et al. (1995) Biochemistry 34, 2809-2814]. This study also raised the possibility of a correlation between ESEEM spectroscopic properties and the nitrogenase phenotype. We now report ESEEM studies on altered MoFe proteins with substitutions at residues α-96-arginine, α-359- arginine, and α-381-phenylalanine to (i) assign the first-shell hydrogen bonding as revealed by the 14N modulation; (ii) explore the mechanistic relevance of the ESEEM signatures to nitrogenase activity; and (iii) study microscopic changes within the polypeptide environment of the FeMo-cofactor. Present ESEEM data reveals that two kinds of 14N modulations are present in wild-type MoFe protein. A new 2-dimensional procedure for high-precision analysis of the ESEEM data of the MoFe proteins shows that the deep wild- type ESEEM modulation (denoted N1) has a hyperfine-coupling constant of A(iso) = 1.05 MHz and nuclear quadrupole coupling parameters of e2qQ = 2.17 MHz, η = 0.59; the other (denoted N2) has a smaller hyperfine coupling of A(iso) = ~0.5 MHz and e2qQ = ~3.5 MHz, η = ~0.4. The N2 ESEEM pattern is more obvious when unmasked by substitutions that result in the loss of the deep N1 modulation. Correlations of the ESEEM properties and catalytic activities of the altered MoFe proteins suggest that (i) the side chain of the α-359-arginine is the source of the deep ESEEM N1 modulation; (ii) one or both of the amide nitrogens of α-356-glycine/α-357-glycine are responsible for the weak N2 modulation; (iii) substitution of the nonpolar α-381-phenylalanine residue, as well as substitution of either the α-195- histidine or α-359-arginine residues, can eliminate the N1 interaction with FeMo-cofactor; and (iv) ESEEM can be used to detect slight reorientations of FeMo-cofactor within its polypeptide pocket, although the mechanistic relevance of the loss or perturbation of the hydrogen-bonding interactions between FeMo-cofactor and polypeptide environment has not yet been established.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Sep 22 1998|
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