ENDOR Spectroscopy Shows Guanine N1 Binds to 4Fe-4S Cluster II of the S-adenosyl methionine-dependent enzyme MoaA - Mechanistic Implications

摘要

The SAM-dependent enzyme MoaA, in concert with MoaC, catalyzes the first step of molybdenum cofactor biosynthesis, the conversion of 5′-GTP into precursor Z. A published X-ray crystal structure of MoaA with the substrate 5′-GTP revealed that the substrate might be binding to the unique iron of one of two 4Fe-4S clusters, through either or both the amino and N1 nitrogen nuclei. 35 GHz CW ENDOR spectroscopy of MoaA with unlabeled and ¹⁵N-labeled substrate and a reduced [4Fe-4S]⁺ cluster now demonstrates that only one nitrogen nucleus is bound to the cluster. Experiments with the substrate analog 5′-ITP further demonstrate that it is the N1 nitrogen that binds. Two of the more distant nitrogen nuclei were also detected by 35 GHz pulsed ENDOR, allowing a rough approximation of their distances from the cluster to be calculated Combining this information with the crystal structure, we have proposed that the guanine base adopts the enol tautomer as N1 binds to Fe4 and the O6-H hydroxyl hydrogen-bonds to S4 of the 4Fe-4S cluster, and that this binding-induced tautomerization may have important mechanistic ramifications.