Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches

CuII‐Containing 1‐Aminocyclopropane Carboxylic Acid Oxidase Is an Efficient Stereospecific Diels–Alderase

“…In most cases the exogenous metal cations have been reported to occupy the active site and coordinate the same amino acids as those that originally coordinated Fe II , namely two histidine and one aspartic acid residues . In particular, Cu II binding has been investigated and Cu II has been shown to bind at the active site of the enzyme with an affinity similar to that of the native Fe II ( K D Fe =4.4 10 −6 m vs. K D Cu =5.5 10 −6 m ) . While the natural Fe II ‐ACCO catalyzes the oxidation of ACC by dioxygen to form the phytohormone ethylene, all its non‐iron reconstituted versions have appeared to be inactive in catalyzing ethylene production .…”

“…[4] Thes ubstitution of the natural Fe II of 1-aminocyclopropane carboxylic acid (ACC) oxidase (Fe II -ACCO) by other metal cations such as Ni II ,C o II ,Z n II ,o rC u II has been reported. [5] In most cases the exogenous metal cations have been reported to occupy the active site and coordinate the same amino acids as those that originally coordinated Fe II , namely two histidine and one aspartic acid residues. [5] In particular,C u II binding has been investigated and Cu II has been shown to bind at the active site of the enzyme with an affinity similar to that of the native Fe II (K DFe = 4.4 10 À6 m vs. K DCu = 5.5 10 À6 m).…”

Artificial Enzymes for Diels‐Alder Reactions

“…[102] Copper(II) was added to apo-1-aminocyclopropane carboxylic acid oxidase (ACCO) instead of the native iron(II). [110] The copper(II)-reconstituted enzyme (ACCO-Cu) was used to catalyze the DA cycloaddition of 8a-c and CP and provided up to quantitative yield and >99 % ee in favor of the endo-9a RR product ( Table 2, entry 12). This unprecedented performance was rationalized by molecular modeling, which suggested that only one coordination geometry of 8a in the active site was possible and that only one path was accessible for CP to react with the bound 8a, thus limiting the number of possible product isomers from four to one (Figure 17).…”

“…have reported on metal‐substitution at the active site of an iron‐containing metalloenzyme to generate a DAase [102] . Copper(II) was added to apo‐1‐aminocyclopropane carboxylic acid oxidase (ACCO) instead of the native iron(II) [110] . The copper(II)‐reconstituted enzyme (ACCO−Cu) was used to catalyze the DA cycloaddition of 8 a – c and CP and provided up to quantitative yield and >99 % ee in favor of the endo ‐ 9a RR product (Table 2, entry 12).…”

The Hunt for the Closed Conformation of the Fruit‐Ripening Enzyme 1‐Aminocyclopropane‐1‐carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study

“…Secondly, we used a copper(II)-reconstituted enzyme with the metal ion as the spectroscopic probe.I ndeed, we previously reported that Cu II binds at the actives ite of ACCO with an affinity similar to that of the native iron ion. [26] We thus reconstituted the singly C60*-labelede nzymew ithC u II and used the RIDME (Relaxation Induced Dipolar Modulation Enhancement)s equence, ap ulsed EPR sequence particularly well suited to determine distances between paramagnetic metal centers and radicals. [27] In this enzyme,t he main Cu-C60* distance distribution is found centered at 2.9 nm with as houlder at 2.0 nm (see Figure S6, Supporting Information).…”