No abstract

(4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two ␣-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An Fe II methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the Fe IV AO bond in the transition state leads to dominant Fe III OO •؊ character, which significantly contributes to the reactivity with either the aromatic -system or the COH -bond. magnetic circular dichroism ͉ density functional calculations ͉ reaction coordinates

Isopenicillin N synthase (IPNS) is a unique mononuclear non-heme Fe enzyme that catalyzes the four electron oxidative double ring closure of its substrate ACV. A combination of spectroscopic techniques including EPR, absorbance, circular dichroism (CD), magnetic CD, and variabletemperature, variable-field MCD (VTVH-MCD) were used to evaluate the geometric and electronic structure of the {FeNO} 7 complex of IPNS coordinated with the ACV thiolate ligand. Density Function Theory (DFT) calculations correlated to the spectroscopic data were used to generate an experimentally calibrated bonding description of the Fe-IPNS-ACV-NO complex. New spectroscopic features introduced by the binding of the ACV thiolate at 13,100 and 19,800 cm −1 are assigned as the NO π*(ip) → Fe d x2−y2 and S π → Fe d x2−y2 charge transfer (CT) transitions, respectively. Configuration interaction mixes S CT character into the NO π*(ip) → Fe d x2−y2 CT transition, which is observed experimentally from the VTVH-MCD data from this transition. Calculations on the hypothetical {FeO 2 } 8 complex of Fe-IPNS-ACV reveal that the configuration interaction present in the {FeNO} 7 complex results in an unoccupied frontier molecular orbital (FMO) with correct orientation and distal O character for H-atom abstraction from the ACV substrate. The energetics of NO/O 2 binding to Fe-IPNS-ACV were evaluated and demonstrate that charge donation from the ACV thiolate ligand renders the formation of the Fe III -superoxide complex energetically favorable, driving the reaction at the Fe center. This single center reaction allows IPNS to avoid the O 2 bridged binding generally invoked in other non-heme Fe enzymes that leads to oxygen insertion (i.e. oxygenase function) and determines the oxidase activity of IPNS.

While iron-bisphosphines have emerged as effective catalysts for C-C cross-coupling, the nature of the in situ formed iron species, elucidation of the active catalysts and the mechanisms of catalysis have remained elusive. A combination of (57)Fe Mössbauer and magnetic circular dichroism (MCD) spectroscopies of well-defined and in situ formed mesityl-iron(II)-SciOPP species combined with density functional theory (DFT) investigations provides the first direct insight into electronic structure, bonding and in situ speciation of mesityl-iron(II)-bisphosphines in the Kumada cross-coupling of MesMgBr and primary alkyl halides using FeCl2(SciOPP). Combined with freeze-trapped solution Mössbauer studies of reactions with primary alkyl halides, these studies demonstrate that distorted square-planar FeMes2(SciOPP) is the active catalyst for cross-coupling and provide insight into the molecular-level mechanism of catalysis. These studies also define the effects of key reaction protocol details, including the role of the slow Grignard addition method and the addition of excess SciOPP ligand, in leading to high product yields and selectivities.