PUB - Publikationen an der Universität Bielefeld

A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules (MoO42-) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoSto(funct)), of only ATP/ADP (MoSto(basal)) and of neither ATP/ADP nor POM clusters (MoSto(zero)), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. V-max was ca. 13 mu mol(phosphate).min(-1).mg(-1) and K-m for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit alpha, inferred from a highly occupied alpha-ATP/Mg2+ and a weaker occupied beta-ATP/no Mg2+-binding site found in the MoSto(funct) structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on V-max, the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoSto(basal) with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis.