Using dysprosium complexes to probe the nitrogenase paramagnetic centers

Abstract

EPR progressive power saturation techniques were used to monitor relaxation enhancement of the nitrogenase paramagnetic centers produced by interaction with Dy3+ complexes. Three models are presented for the relationship between the degree of enhancement and the distance of closest approach of Dy3+ to the intrinsic metal cluster. In the first model, the perturbing dysprosium ions are represented as a single average site. The second and third models are variations of the treatment of Innes and Brudvig [(1989) Biochemistry 28, 1116-1125] and assume the unknown protein to be spherical with Dy3+ dispersed either randomly over the surface of the protein or randomly in solution. Using these models, the distance of closest approach of the Dy3+ complex to the [4Fe-4S] cluster in the Fe-protein from Azotobacter vinelandii was determined to be 5.0-6.5 A. Similarly, the distance of closest approach to FeMoco in the MoFe-protein was determined to be 0.0-1.2 A, which, when corrected to the fact that FeMoco exists as an S = 3/2 spin state, indicates that the distance is > or = 6 A. These distances did not change when (1) either protein was in the presence of the other, (2) both proteins were cross-linked to each other, or (3) the Fe-protein from A. vinelandii was mixed with the MoFe-protein from Clostridium pasteurianum. On the other hand, formation of the inactive complex of the Fe-protein from C. pasteurianum with the MoFe-protein from A. vinelandii blocked dysprosium-induced relaxation enhancement, implying that each component protein overlaps the metal cluster in the complementing protein.