Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of NifEN-bound precursor and...

NifEN plays a key role in the biosynthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase. A scaffold protein that hosts the conversion of a FeMoco precursor to a mature cofactor, NifEN can assume three conformations during the process of FeMoco maturation. One, designated ΔnifB NifEN, contains only two permanent [Fe(4)S(4)]-like clusters. The second, designated NifEN(Precursor), contains the permanent clusters and a precursor form of FeMoco. The third, designated NifEN("FeMoco"), contains the permanent [Fe(4)S(4)]-like clusters and a fully complemented, "FeMoco"-like structure. Here, we report a variable-temperature, variable-field magnetic circular dichroism spectroscopic investigation of the electronic structure of the metal clusters in the three forms of dithionite-reduced NifEN. Our data indicate that the permanent [Fe(4)S(4)]-like clusters are structurally and electronically conserved in all three NifEN species and exhibit spectral features of classic [Fe(4)S(4)](+) clusters; however, they are present in a mixed spin state with a small contribution from the S > ½ spin state. Our results also suggest that both the precursor and "FeMoco" have a conserved Fe/S electronic structure that is similar to the electronic structure of FeMoco in the MoFe protein, and that the "FeMoco" in NifEN("FeMoco") exists, predominantly, in an S = 3/2 spin state with spectral parameters identical to those of FeMoco in the MoFe protein. These observations provide strong support to the outcome of our previous EPR and X-ray absorption spectroscopy/extended X-ray absorption fine structure analysis of the three NifEN species while providing significant new insights into the unique electronic properties of the precursor and "FeMoco" in NifEN.