The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS − )-, sulfenate (RSO − )-, and sulfinate (RSO 2 − )-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO − species changes upon protonation as the S-O bond is elongated (by ~0.1 Å). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe III in the active site of NHase as CysS − , CysSOH, and CysSO 2 − both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z eff of the Fe and reveals that the Fe in [FeNO] 6 NHase species has a Z eff very similar to that of its photolyzed Fe III counterpart. DFT calculations reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts significant charge from the formally t 2 6 low-spin metal to the coordinated NO.

Bleomycin (BLM), a glycopeptide antibiotic chemotherapy agent, is capable of single- and double-strand DNA damage. Activated bleomycin (ABLM), a low-spin Fe(III)-OOH complex, is the last intermediate detected prior to DNA cleavage following hydrogen-atom abstraction from the C-4' of a deoxyribose sugar moiety. The mechanism of this C-H bond cleavage reaction and the nature of the active oxidizing species are still open issues. We have used kinetic measurements in combination with density functional calculations to study the reactivity of ABLM and the mechanism of the initial attack on DNA. Circular dichroism spectroscopy was used to directly monitor the kinetics of the ABLM reaction. These experiments yield a deuterium isotope effect, kH/kD approximately 3 for ABLM decay, indicating the involvement of a hydrogen atom in the rate-determining step. H-atom donors with relatively weak X-H bonds accelerate the reaction rate, establishing that ABLM is capable of hydrogen-atom abstraction. Density functional calculations were used to evaluate the two-dimensional potential energy surface for the direct hydrogen-atom abstraction reaction of the deoxyribose 4'-H by ABLM. The calculations confirm that ABLM is thermodynamically and kinetically competent for H-atom abstraction. The activation and reaction energies for this pathway are favored over both homolytic and heterolytic O-O bond cleavage. Direct H-atom abstraction by ABLM would generate a reactive Fe(IV)=O species, which would be capable of a second DNA strand cleavage, as observed in vivo. This study provides experimental and theoretical evidence for direct H-atom abstraction by ABLM and proposes an attractive mechanism for the role of ABLM in double-strand cleavage.

Tyrosine Hydroxylase (TH) is a pterin-dependent non-heme iron enzyme that catalyzes the hydroxylation of L-tyr to L-DOPA in the rate-limiting step of catecholamine neurotransmitter biosynthesis. We have previously shown that the Fe II site in Phenylalanine Hydroxylase (PAH) converts from 6C to 5C only when both substrate + cofactor are bound. However, steady-state kinetics indicate that TH has a different cosubstrate binding sequence (pterin + O 2 + L-tyr) than PAH (L-phe + pterin + O 2 ). Using x-ray absorption spectroscopy (XAS), and variable-temperature-variable-field magnetic circular dichroism (VTVH MCD) spectroscopy, we have investigated the geometric and electronic structure of the WT TH and two mutants, S395A and E332A, and their interactions with substrates. All three forms of TH undergo 6C → 5C conversion with tyr + pterin, consistent with the general mechanistic strategy established for O 2 -activating non-heme iron enzymes. We have also applied single-turnover kinetic experiments with spectroscopic data to evaluate the mechanism of the O 2 and pterin reactions in TH. When the Fe II site is 6C, the two-electron reduction of O 2 to peroxide by Fe II and pterin is favored over individual one-electron reactions, demonstrating that both a 5C Fe II and a redox-active pterin are required for coupled O 2 reaction. When the Fe II is 5C, the O 2 reaction is accelerated by at least 2 orders of magnitude. Comparison of the kinetics of WT TH, which produces Fe IV =O + 4a-OH-pterin, and E332A TH, which does not, shows that the E332 residue plays an important role in directing the protonation of the bridged Fe II -OO-pterin intermediate in WT to productively form Fe IV =O, which is responsible for hydroxylating L-tyr to L-DOPA.