SPring-8, the large synchrotron radiation facility

Release Date

05 Apr, 2013

Background
Class Ia ribonucleotide reductase (RR) and soluble methane monooxygenase (sMMO) perform thermodynamically challenging H atom abstraction from tyrosine and hydroxylation of methane by generating highly reactive Fe(III)Fe(IV) and Fe(IV)₂ intermediates, respectively. For these transient intermediate species, various structures have been suggested including an {Fe2(μO)₂} diamond core, a protonated diamond core, and a mono-oxo bridged core, but consensus has not been reached. Their structural elucidation is essential for understanding their reaction mechanisms on a molecular level with consequent medical and industrial applications.

Vibrational spectroscopy can provide valuable information on the geometric structures of transient species, but traditional techniques such as resonance Raman (rR) spectroscopy have not been successful due to the photolability of the intermediates. Therefore, nuclear resonance vibrational spectroscopy (NRVS) is the ideal alternative, as it probes all the vibrational modes that contain significant Fe displacement with very bright, tunable X-rays at 14.4 keV.

To establish a basis to correlate NRVS spectral features with the geometric structures of high-valent diiron species, four structurally characterized synthetic compounds, mono-oxo and di-oxo bridged Fe(III)Fe(IV) and Fe(IV)₂ complexes have been characterized using NRVS in combination with density functional theory (DFT) calculations, with evaluations of the effects of variations in the bridging structure and Fe oxidation and spin states. A library of DFT-simulated NRVS spectra for the possible structures of Q has also been generated to confirm the capability of NRVS to distinguish the structural candidates and to set up application to Q in sMMO.

Achievements
Structurally well-established Fe(III)Fe(IV) and Fe(IV)₂ model complexes, containing mono-oxo and di-oxo diamond cores (Fig. 1), have been characterized using NRVS. They display intense features in the energy region below 450 cm^-1, which are insensitive to variations in Fe oxidation state while very sensitive to Fe spin state and importantly the bridging structure of the Fe₂ core. NRVS spectra obtained from the low-spin model complexes reveal that while the mono-oxo species display three broad bands in the energy region below 450 cm^-1, the di-oxo species show a five-peak pattern, as the in-plane translational and rotational motions of the Fe₂ core bisect bonds in the mono-oxo species (Fig. 2). However, DFT calculations and NRVS data of relevant high-spin species show that upon low- to high-spin conversion of Fe, the σ-antibond increases and the π-antibonding character decreases. Thus, the high-spin mono-oxo species displays a more split peak pattern than the high-spin di-oxo species (Fig. 3). Using the same DFT methodology, NRVS spectra have been calculated for intermediate Q using the range of structures proposed up to date. These predicted spectra show that NRVS should be able to distinguish among these structural candidates with high sensitivity to variations in bridging structure; a structure with a mono-oxo bridge would show multiple peaks with comparable intensities, while a diamond core structure would display a major intense peak with significantly decreased additional contributions (from carboxylate bridge bends (noted with asterisks))

Future Developments
Correlations between the NRVS spectra and geometric structures of high-valent diiron complexes observed in this study will be extended to determine the geometric structures of transient but trapped, active intermediates X and Q. The structural elucidation of these key intermediates in RR and sMMO will clarify the mechanisms of these enzymes in performing the thermodynamically challenging O-H and C-H bond cleavages, respectively.

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