Aluminum fluoride inhibition of nitrogenase: stabilization of a nucleotide.Fe-protein.MoFe-protein complex.
Coupling of ATP hydrolysis to electron transfer in nitrogenase has properties similar to nucleotide-dependent switch proteins. Aluminum fluoride, a powerful inhibitor of some switch proteins, is a progressive, slowly reversible (t1/2 for reversal > 21 h) inhibitor of nitrogenase that requires both component proteins (Fe-protein and MoFe-protein) and nucleotide (either ATP or ADP). The pseudo first-order inhibition is dependent on the aluminum fluoride species, AlF4, and is linear with [Al] concentration (nonsaturating) at a pH optimum near 7.1-7.3. The inhibitor appears to react with the transient complex of the two component proteins and nucleotide. Although ADP can support the AlF inhibition, the rate of inhibition is more than 30-fold greater with ATP, which suggests the reactive conformation more closely resembles ATP hydrolysis. Conditions that increase enzymic turnover (protein concentration and component ratio) also increase the rate of inhibition, while ionic strength which slows enzymic activity spares the inhibition. The inhibited protein was isolated by gel filtration chromatography and found to be an AlF4-ADP-Fe-protein.MoFe-protein complex with the ratio of 2:1 that is consistent with two active sites per MoFe-protein alpha 2 beta 2 tetramer. Hence, inhibition by AlF4 is the stabilization of a complex that no longer hydrolyzes ATP or reduces substrates. We propose that AlF-ADP is tightly bound only in Fe-protein conformations obtained in the complex with MoFe-protein. Ligands (including Arg-46) at the base of a flexible flap on the Fe-protein could immobilize MoFe-protein--Fe-protein interface, thereby preventing dissociation of the complex.
Renner
K A
KA
Department of Biochemistry, University of Minnesota, Minneapolis 55455, USA.
Howard
J B
JB
D000227:Adenine Nucleotides / Q000737:chemistry; D017607:Aluminum Compounds / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005459:Fluorides / Q000494:pharmacology*; D008667:Metalloproteins / Q000737:chemistry; D008958:Models, Molecular; D009591:Nitrogenase / Q000037:antagonists & inhibitors*; D011487:Protein Conformation
D000227:Adenine Nucleotides; D017607:Aluminum Compounds; D004791:Enzyme Inhibitors; D008667:Metalloproteins; D009591:Nitrogenase; D005459:Fluorides; C032311:aluminum fluoride