Metal-Ion Valencies of the FeMo Cofactor in CO-Inhibited and Resting State Nitrogenase by ⁵⁷Fe Q-Band ENDOR

Abstract

The resting state of nitrogenase shows an S = 3/2 electron paramagnetic resonance (EPR) signal resulting from the FeMo-cofactor (M-center; inorganic portion, [Mo, Fe7, S9]) of the MoFe-protein. When the enzyme undergoes turnover under a CO atmosphere, this signal disappears and two new ones appear: one under low pressure of CO (denoted lo-CO; 0.08 atm) with g = [2.09, 1.97, 1.93] and the other under high pressure of CO (denoted hi-CO; 0.5 atm) with g = [2.06, 2.06, 2.17]. Our recent Q-band (35 GHz) 57Fe and 13C electron nuclear double resonance (ENDOR) studies clearly identified [FeMo-cofactor][CO] n , as the origin of the EPR signals from both lo-CO (n = 1) and hi-CO (n = 2) [Christie, P. D.; Lee, H. I.; Cameron, L. M.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 8707−8709 and Pollack, R. C.; Lee, H. I.; Cameron, L. M.; Derose, V. J.; Hales, B. J.; Orme-Johnson, W. H.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117, 8686−8687], and a previous paper discusses CO binding in detail [Lee, H. I.; Cameron, L. M.; Hales, B. J.; Hoffman, B. M. J. Am. Chem. Soc. 1997, 119, 10121−10126]. We now present complete orientation-selective 57Fe ENDOR measurements of the CO-bound FeMo-cofactor in both lo- and hi-CO forms of the MoFe-protein from Azotobacter vinelandii. The 57Fe ENDOR signals associated with the seven Fe ions of the FeMo-cofactor of lo-CO can be completely assigned and interpreted in terms of four magnetically distinct iron signals. Analysis of these signals following the procedures of Mouesca et al. [Mouesca, J.-M.; Noodleman, L.; Case, D. A.; Lamotte, B. Inorg. Chem. 1995, 34, 4347−4359] has led us to propose valence assignments and charges for the cofactor cluster, [Mo, Fe7, S9]+ = [Mo4+, Fe3+ 1, Fe2+ 6, S2- 9]+, organized into one Fe2.5+ pair and five Fe2+ ions, [Mo4+, (2Fe2.5+)1, Fe2+ 5, S2- 9]+. The result is a formal d-electron count of 43. ENDOR and functional studies indicate that the lo-CO, hi-CO, and resting states of the M-center are all at the same oxidation level. Hence, the proposed valency assignments apply to all three states.

Biosynthesis of the Metalloclusters of Molybdenum Nitrogenase

“…The isolated FeMoco has been shown to be anionic (4), yet the proposed core charge of FeMoco in the resting state is ϩ1 or ϩ3 (37,55). The overall negative charge of the cofactor, therefore, is believed to originate from its endogenous homocitrate moiety, which is Ϫ4 if the -OH group is deprotonated.…”

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry

“…These methods contribute detailed information of all of the types listed in Table 1, but most importantly do so not only for resting-state metal centers, but also for reactive intermediates (as we shall see), which seldom are amenable to x-ray diffraction methods (15) (17), and perhaps most dramatically, the catalytic molybdenum-iron cofactor of nitrogenase (14). However, it includes the other types of information of Table 1, too, ranging from the location of a single electron (hole) and a single proton in a highvalent intermediate (18), a task for which ENDOR spectroscopy is perhaps uniquely suited, to information about dynamics (19), an area where ENDOR͞ESEEM spectroscopies might have been thought unsuitable.…”

“…Thus, with proper isotopic labeling it is possible to characterize every atom associated with a paramagnetic center (5,13); because the electron spin is the ''detector,'' the methods are more sensitive than conventional NMR and see ''nonstandard'' nuclei (e.g., Fe resonances from one metal cluster without interference from other clusters, as in nitrogenase (14) and from a cluster in the presence of excess inorganic Fe (see below). The obvious limitation to paramagnetic resonance methods, of course, is that they require a paramagnetic center, be it metal ion or radical.…”

Ammonia synthesis at low temperatures

“…͑2͒. 3,4 The active site of the enzyme is very well characterized, [20][21][22][23][24][25][26][27][28][29][30][31][32] but the detailed molecular mechanism is not as well established as for the metal surface process. It is generally believed that the biological process does not involve initial breaking of the N-N bond, 33 and recent DFT calculations on different Mo, Fe sulfide complexes modeling the FeMoco 34-36 support this picture.…”