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The hydrolytic properties of the mutant ␣ 3 (␤T165S) 3 ␥ and wild-type ␣ 3 ␤ 3 ␥ subcomplexes of TF 1 have been compared. Whereas the wild-type complex hydrolyzes 50 M ATP in three kinetic phases, the mutant complex hydrolyzes 50 M ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg 2؉ , the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0.1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnoverdependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg 2؉ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg

Methanobactins (Mbns) are a family of copper-binding peptides involved in copper uptake by methanotrophs, and are potential therapeutic agents for treating diseases characterized by disordered copper accumulation. Mbns are produced via modification of MbnA precursor peptides at cysteine residues catalyzed by the core biosynthetic machinery containing MbnB, an iron-dependent enzyme, and MbnC. However, mechanistic details underlying the catalysis of the MbnBC holoenzyme remain unclear. Here, we present crystal structures of MbnABC complexes from two distinct species, revealing that the leader peptide of the substrate MbnA binds MbnC for recruitment of the MbnBC holoenzyme, while the core peptide of MbnA resides in the catalytic cavity created by the MbnB–MbnC interaction which harbors a unique tri-iron cluster. Ligation of the substrate sulfhydryl group to the tri-iron center achieves a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complexes together with functional investigation of MbnB variants identified a conserved catalytic aspartate residue as a general base required for MbnBC-mediated MbnA modification. Together, our study reveals the similar architecture and function of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzymes.

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Addition of Al3+ and F- to the alpha3beta3gamma subcomplex of the TF1-ATPase containing MgADP in one catalytic site causes slow, complete inactivation as the ADP-fluoroaluminate complex is formed. This conflicts with the "bisite" stochastic model suggested earlier (Issartel, J. P., Dupuis, A., Lunardi, J. & Vignais, P. V. (1991) Biochemistry 30, 4726-4733] on the finding that complete inactivation of the bovine mitochondrial F1-ATPase by Al3+, F-, Mg2+, and excess ADP occurs as ADP-fluoroaluminate complexes form in two catalytic sites. When Al3+ and F- were added to alpha3beta3gamma containing MgADP in two catalytic sites, inactivation accelerated 8-fold, indicating catalytic to catalytic site cooperativity. When added to alpha3beta3gamma containing MgADP bound to one or two catalytic sites prior to addition of Al3+ and F-, phosphate inhibits formation of the ADP-fluoroaluminate complex. When introduced after adding 200 microM ADP plus Mg2+ to alpha3beta3gamma, but before adding Al3+ and F-, phosphate accelerated formation of the ADP-fluoroaluminate complex 3-fold. Sulfite accelerated formation of the ADP-fluoroaluminate complex 9-fold when 200 microM ADP plus Mg2+ was added to alpha3beta3gamma before adding Al3+ and F-. The accelerations induced by phosphate or sulfite in the presence of excess ADP and Mg2+ suggest noncatalytic to catalytic site cooperativity. When Al3+ and F- were added to the (alphaD261N)3beta3gamma subcomplex containing MgADP in a single catalytic site, the ADP-fluoroaluminate complex formed at least 10-fold more slowly than observed with wild-type under the same conditions. Therefore, the catalytic site containing MgADP recognizes the alphaD261N substitution when noncatalytic sites are empty. Cross-linking alpha to gamma or beta to gamma by oxidizing the (alphaA396C)3beta3(gammaA22C) and alpha3(betaD390C)3(gammaS90C) subcomplexes, respectively, abolishes cooperative formation of ADP-fluoroaluminate complexes in two catalytic sites. ADP-fluoroaluminate complex formation is restricted to a single catalytic site in the oxidized double mutants. The alpha3beta3delta subcomplex does not form an inhibitory ADP-fluoroaluminate complex under any of the conditions examined for the alpha3beta3gamma subcomplexes.