Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study changes in the polypeptide environment of the FeMo-cofactor that were elicited by amino-acid substitutions within the nitrogenase MoFe protein a-subunit. A previous ESEEM study [Thomann et al. (1991) Proc. Natl. Acad. Sci. U.SA. 88, 6620] detected modulation arising from nitrogen coupled to the S = 3/2 spin system of the FeMo-cofactor (Fe7S9Mo:homocitrate). Such modulation was found to be sensitive to the substitution of α-195His by α-195Asn as indicated by whole-cell ESEEM analysis of mutant strains from Azotobacter vinelandii. Subsequent structural studies revealed that the α-195His residue does not provide direct N-coordination to the cluster but is within hydrogen-bonding distance of one of a set of three sulfides that bridge the FeMo-cofactor subcluster fragments. In the present work, the ESEEM analysis is extended to both partially purified α-195Asn MoFe protein and purified MoFe protein from an additional mutant strain in which α-195His is replaced by α-195Gln. The dramatic decrease in the intensity of the ESEEM signal resulting from the α-195Asn substitution in whole cells was confirmed for the case of the isolated α-195Asn MoFe protein. In contrast, substitution of β-195His by α-195Gln caused no detectable change in the modulation. Simulations of the α-195His and α-195Gln ESEEM data give quadrupole parameters of e2qQ = 2.2 MHz and η = 0.5. Glutamine and histidine have similar chain lengths from the a-carbon to the protonated nitrogen that could provide a hydrogen bond to the FeMo-cofactor, whereas asparagine is shorter by one C-C bond and, therefore, cannot provide the putative hydrogen bond to the FeMo-cofactor under the constraints of the current structural models. However, simulations show that the ESEEM is quite sensitive to the electronic parameters of the 14N nuclei, and therefore it is highly unlikely that an identical nitrogen modulation could arise from both an imidazole ring nitrogen provided by α-195His and the glutamine amide group provided by α-195Gln. Thus, these results indicate that the observed nitrogen modulation is not directly associated with the hydrogen bond provided by α-195His but rather with the nitrogen moiety of a different residue whose proximity to the FeMo-cofactor is sensitive to certain substitutions at the α-195His position. The ESEEM data, which provide a delicate probe of the local structure of the FeMo-cofactor, do however strongly suggest that either α-195His or α-195Gln, but not α-195Asn, provides a hydrogen bond to FeMo-cofactor. This interpretation is in line with biochemical characterizations showing that both α-195His and α-195Gln MoFe proteins can bind N2, whereas the α-195Asn MoFe protein cannot. Thus, this information provides a correlation of phenotypic, biochemical, and spectroscopic properties of altered MoFe proteins produced by site-directed mutagenesis and should be useful in assigning mechanistic importance to specific structural features of the MoFe protein.
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