Scientific Report 2006

Molecular Biology

Quantum Chemistry of Redox-Active Metalloenzymes

L. Noodleman, D.A. Case, W.-G. Han, V. Pelmenschikov, J.A. Fee, L. Hunsicker-Wang,* T. Lovell,** T. Liu***

* Trinity University, San Antonio, Texas
** AstraZeneca R&D, Mölndal, Sweden
*** University of Maryland, College Park, Maryland

We use a combination of modern quantum chemistry (density functional theory, DFT) and classical electrostatics to describe the energetics, reaction pathways, and spectroscopic properties of metalloenzymes.

Critical biosynthetic and regulatory processes may involve catalytic transformations of fairly small molecules or groups by transition-metal centers. The iron-molybdenum cofactor center of nitrogenase catalyzes the multielectron reduction of molecular nitrogen to 2 molecules of ammonia plus molecular hydrogen. We are continuing our work on the catalytic cycle of this enzyme, following up on our earlier work on the structure and oxidation state of the cofactor complex in the “resting enzyme” before multielectron reduction and nitrogen binding.

On the basis of DFT calculated vs experimental physical properties, including redox potentials, cluster geometries, and Mössbauer isomer shifts, the core cluster has a MoFe₇S₉X prismane active site, where the central X most likely is nitride and the “resting cluster oxidation state” is Mo(IV)Fe(II)₄Fe(III)₃. If the central ligand is nitride, as we have proposed, this ligand is not a substrate or a reaction product of the catalytic cycle. Instead, nitride is inserted into a central vacancy site of a more open iron-molybdenum cofactor precursor, probably in a noncatalytic deamination process that occurs before insertion of the cluster into the iron-molybdenum protein.

Class I ribonucleotide reductases are aerobic enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides, providing the required building blocks for DNA replication and repair. These ribonucleotide-to-deoxyribonucleotide reactions occur via a long-range radical (or proton-coupled electron transfer) propagation mechanism initiated by a fairly stable tyrosine radical, “the pilot light.” When this pilot light goes out, the tyrosine radical is regenerated by a high-oxidation-state Fe(III)-Fe(IV)-oxo enzyme called intermediate X. We are using DFT and electrostatics calculations in combination with analysis of Mössbauer, electron nuclear double resonance, and magnetic circular dichroism spectroscopies to search for a proper structural and electronic model for intermediate X.

We have also examined the mechanism of formation of intermediate X, starting from an earlier Fe(III)₂-μ-peroxo intermediate (Fig. 1). Spectroscopic and quantum chemical DFT evidence indicates that the formation of intermediate X is proton catalyzed. On the basis of calculations of spectroscopic parameters and energies, we propose that intermediate X contains a dioxo bridging the Fe(III)-Fe(IV) in an asymmetric diamond structure. The Fe(IV) site is farther from and the Fe(III) site is closer to the redox-active tyrosine 122. Figure 2 shows the molecular orbitals corresponding to the lowest energy Fe(IV) d→d optical excitation. The 3 Fe(IV) d→d bands that we predict on the basis of DFT vertical self-consistent reaction field methods are in excellent agreement with the bands observed by using magnetic circular dichroism spectroscopy. Further exploration of the tyrosine radical activation and subsequent catalytic cycle are planned.

Fig. 1. A feasible path showing how ribonucleotide reductase intermediate X is formed by the reaction of oxygen with the reduced ribonucleotide reductase–R2 di-iron center. Reproduced with permission from J. Am. Chem. Soc. 127:15778, 2005. Copyright 2005 American Chemical Society.

Fig. 2. Our proposed model for the active site of class I ribonucleotide reductase intermediate X. Molecular orbital plots show the lowest energy Fe(IV) d→d optical excitation.

Publications

Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Active site structure of class I ribonucleotide reductase intermediate X: a density functional theory analysis of structure, energetics, and spectroscopy. J. Am. Chem. Soc. 127:15778, 2005.

Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Density functional theory study of Fe(IV) d-d optical transitions in active-site models of class I ribonucleotide reductase intermediate X with vertical self-consistent reaction field methods. Inorg. Chem. 45:8533, 2006.

Han, W.-G., Liu, T., Lovell, T., Noodleman, L. DFT calculations of ⁵⁷Fe Mössbauer isomer shifts and quadrupole splittings for iron complexes in polar dielectric media: applications to methane monooxygenase and ribonucleotide reductase. J. Comput. Chem. 27:1292, 2006.

Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Seven clues to the origin and structure of class-I ribonucleotide reductase intermediate X. J. Inorg. Biochem. 100:771, 2006.

Noodleman, L., Han, W.-G. Structure, redox, pK_a, spin: a golden tetrad for understanding metalloenzyme energetics and reaction pathways. J. Biol. Inorg. Chem. 11:674, 2006.