ELECTRON-SPIN ECHO ENVELOPE MODULATION SPECTROSCOPIC ANALYSIS OF ALTERED NITROGENASE MOFE PROTEINS FROM AZOTOBACTER-VINELANDII

Electron spin echo envelope modulation (ESEEM) spectroscopy was used t o study changes in the polypeptide environment of the FeMo-cofactor th at were elicited by amino-acid substitutions within the nitrogenase Mo Fe protein alpha-subunit. A previous ESEEM study [Thomann et al. (1991 ) Proc. Natl. Acad, Sci, U.S.A. 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 th e substitution of alpha-195(His) by alpha-195(Asn) as indicated by who le-cell ESEEM analysis of mutant strains from Azotobacter vinelandii. Subsequent structural studies revealed that the alpha-195(His) residue does not provide direct N-coordination to the cluster but is within h ydrogen-bonding distance of one of a set of three sulfides that bridge the FeMo-cofactor subcluster fragments. In the present work, the ESEE M analysis is extended to both partially purified alpha-195(Asn) MoFe protein and purified MoFe protein from an additional mutant strain in which alpha-195(His) is replaced by alpha-195(Gln). The dramatic decre ase in the intensity of the ESEEM signal resulting from the alpha-195( Asn) substitution in whole cells was confirmed for the case of the iso lated alpha-195(Asn) MoFe protein. In contrast, substitution of alpha- 195(His) by alpha-195(Gln) caused no detectable change in the modulati on. Simulations of the alpha-195(His) and alpha-195(Gln) ESEEM data gi ve quadrupole parameters of e(2)qQ = 2.2 MHz and eta = 0.5. Glutamine and histidine have similar chain lengths from the cc-carbon to the pro tonated nitrogen that could provide a hydrogen bond to the FeMo-cofact or, whereas asparagine is shorter by one C-C bond and, therefore, cann ot provide the putative hydrogen bond to the FeMo-cofactor under the c onstraints of the current structural models, However, simulations show that the ESEEM is quite sensitive to the electronic parameters of the N-14 nuclei, and therefore it is highly unlikely that an identical ni trogen modulation could arise from both an imidazole ring nitrogen pro vided by alpha-195(His) and the glutamine amide group provided by alph a-195(Gln). Thus, these results indicate that the observed nitrogen mo dulation is not directly associated with the hydrogen bond provided by alpha-195(His) but rather with the nitrogen moiety of a different res idue whose proximity to the FeMo-cofactor is sensitive to certain subs titutions at the alpha-195(His) position, The ESEEM data, which provid e a delicate probe of the local structure of the FeMo-cofactor, do how ever strongly suggest that either alpha-195(His) or alpha-195(Gln), bu t not alpha-195(Asn), provides a hydrogen bond to FeMo-cofactor, This interpretation is in line with biochemical characterizations showing t hat both alpha-195(His) and alpha-195(Gln) MoFe proteins can bind Nz w hereas the alpha-195(Asn) MoFe protein cannot. Thus, this information provides a correlation of phenotypic, biochemical, and spectroscopic p roperties of altered MoFe proteins produced by site-directed mutagenes is and should be useful in assigning mechanistic importance to specifi c structural features of the MoFe protein.