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. 2007 Jun 13;129(23):7427-38.
doi: 10.1021/ja071364v. Epub 2007 May 17.

VTVH-MCD and DFT studies of thiolate bonding to [FeNO]7/[FeO2]8 complexes of isopenicillin N synthase: substrate determination of oxidase versus oxygenase activity in nonheme Fe enzymes

Christina D Brown  1 , Michael L Neidig, Matthew B Neibergall, John D Lipscomb, Edward I Solomon
Affiliations

VTVH-MCD and DFT studies of thiolate bonding to [FeNO]7/[FeO2]8 complexes of isopenicillin N synthase: substrate determination of oxidase versus oxygenase activity in nonheme Fe enzymes

Christina D Brown et al. J Am Chem Soc. .

Abstract

Isopenicillin N synthase (IPNS) is a unique mononuclear nonheme 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 variable-temperature, 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 pi*(ip) --> Fe dx2-y2 and S pi--> Fe dx2-y2 charge transfer (CT) transitions, respectively. Configuration interaction mixes S CT character into the NO pi*(ip) --> Fe dx2-y2 CT transition, which is observed experimentally from the VTVH-MCD data from this transition. Calculations on the hypothetical {FeO2}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/O2 binding to Fe-IPNS-ACV were evaluated and demonstrate that charge donation from the ACV thiolate ligand renders the formation of the FeIII-superoxide complex energetically favorable, driving the reaction at the Fe center. This single center reaction allows IPNS to avoid the O2 bridged binding generally invoked in other nonheme Fe enzymes that leads to oxygen insertion (i.e., oxygenase function) and determines the oxidase activity of IPNS.

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Figures

Figure 1
Figure 1
Temperature-dependent EPR Spectra of Fe-IPNS-ACV-NO and Boltzmann fit to the Curie law of relative EPR intensities to give an axial ZFS value of D = 12.5 ± 0.5cm−1 (inset).
Figure 2
Figure 2
Absorbance (10K, top), CD (5K, middle) and MCD (5K,7T, bottom) spectra collected for Fe-IPNS-ACV-NO with Gaussian best fits. Comparison to the MCD spectrum of Fe-EDTA-NO (inset) reveals two new spectral features seen in the spectra upon addition of ACV to Fe-IPNS-NO (arrows).
Figure 3
Figure 3
Fe-IPNS-ACV-NO VTVH MCD data collected at 13,260cm−1 (left) and 22,730cm−1 (right) demonstrate different nesting behaviors. (Arrows indicate energy where the isotherms were collected.)
Figure 4
Figure 4
UBP86+10%HF/6–311G*/6–31G* Optimized Fe-IPNS-ACV-NO complex.
Figure 5
Figure 5
Contours of the lowest unoccupied α and β molecular orbitals from the ground state wavefunction of Fe-IPNS-ACV-NO.
Figure 6
Figure 6
Molecular orbital diagram of Fe-IPNS-ACV-NO. The 5 lowest unoccupied β orbitals are comprised of mostly Fe d character, while the 2 lowest unoccupied α orbitals are comprised of mostly NOπ* character, consistent with an electronic structure description of a high-spin FeIII antiferromagnetically coupled to NO−.
Figure 7
Figure 7
ΔSCF charge transfer transition for the Fe-IPNS-ACV-NO complex calculated using ADF/BP86/TZV with Zeff = 25.6.
Figure 8
Figure 8
Contours of the lowest unoccupied α and β molecular orbitals from the ground state wavefunction of Fe-IPNS-ACV-O2
Figure 9
Figure 9
Molecular orbital diagram of Fe-IPNS-ACV-O2. The 5 lowest unoccupied β orbitals are comprised of mostly Fe d character, while the lowest unoccupied α orbital is comprised of mostly O2 π* character, consistent with an electronic structure description of a high-spin FeIII antiferromagnetically coupled to O2.−.
Figure 10
Figure 10
Energy profiles of NO/O2 binding to Fe-IPNS-ACV and Fe-PAH.
Figure 11
Figure 11
Fe-IPNS-ACV-O2 FMO with proper orientation for H-atom abstraction of cysteine β-methylene H from ACV.
Figure 12
Figure 12
Comparison of plausible intermediates for mononuclear non-heme Fe oxygenases (αKG-dependent dioxygenases, shown top) and oxidases (IPNS, shown bottom).
Scheme 1
Scheme 1
IPNS catalyzes a four electron oxidative double ring closure of ACV to form isopenicillin N.
See this image and copyright information in PMC

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