Axial Ligand Effects on Vibrational Dynamics of Iron in Heme Carbonyl Studied by Nuclear Resonance Vibrational Spectroscopy

Nuclear resonance vibrational spectroscopy (NRVS) and density functional theory calculation (DFT) have been applied to illuminate the effect of axial ligation on the vibrational dynamics of iron in heme carbonyl. The analyses of the NRVS data of five- (5c) and six-coordinate (6c) heme–CO complexes indicate that the prominent feature of ⁵⁷Fe partial vibrational density of state (⁵⁷FePVDOS) at the 250–300 cm^–1 region is significantly affected by the association of the axial ligand. The DFT calculations predict that the prominent ⁵⁷FePVDOS is composed of iron in-plane motions which are coupled with porphyrin pyrrole in-plane (ν₄₉, ν₅₀, and ν₅₃), an out-of-plane (γ₈) (two of four pyrrole rings include the in-plane modes, while the rest of pyrrole rings vibrate along the out-of-plane coordinate), and out-of-phase carbonyl C and O atom displacement perpendicular to the Fe–C–O axis. Thus, in the case of the 5c CO–heme the prominent ⁵⁷FePVDOS shows sharp and intense feature because of the degeneracy of the <i>e</i> symmetry mode within the framework of <i>C</i>_4<i>v</i> symmetry molecule, whereas the association of the axial imidazole ligand in the 6c complex with the lowered symmetry results in split of the degenerate vibrational energy as indicated by broader and lower intensity features of the corresponding NRVS peak compared to the 5c structure. The vibrational energy of the iron in-plane motion in the 6c complex is higher than that in 5c, implying that the iron in the 6c complex includes stronger in-plane interaction with the porphyrin compared to 5c. The iron in-plane mode above 500 cm^–1, which is predominantly coupled with the out-of-phase carbonyl C and O atom motion perpendicular to Fe–C–O, called as Fe–C–O bending mode (δ_Fe–C–O), also suggests that the 6c structure involves a larger force constant for the <i>e</i> symmetry mode than 5c. The DFT calculations along with the NRVS data suggest that the stiffened iron in-plane motion in the 6c complex can be ascribed to diminished pseudo-Jahn–Teller instability along the <i>e</i> symmetry displacement due to an increased <i>a</i>₁–<i>e</i> orbital energy gap caused by σ* interaction between the iron d_<i>z</i>² orbital and the nitrogen p orbital from the axial imidazole ligand. Thus, the present study implicates a fundamental molecular mechanism of axial ligation of heme in association with a diatomic gas molecule, which is a key primary step toward versatile biological functions

Nuclear Resonance Vibrational Spectroscopic Definition of Peroxy Intermediates in Nonheme Iron Sites

Fe^III-(hydro)­peroxy intermediates have been isolated in two classes of mononuclear nonheme Fe enzymes that are important in bioremediation: the Rieske dioxygenases and the extradiol dioxygenases. The binding mode and protonation state of the peroxide moieties in these intermediates are not well-defined, due to a lack of vibrational structural data. Nuclear resonance vibrational spectroscopy (NRVS) is an important technique for obtaining vibrational information on these and other intermediates, as it is sensitive to all normal modes with Fe displacement. Here, we present the NRVS spectra of side-on Fe^III-peroxy and end-on Fe^III-hydroperoxy model complexes and assign these spectra using calibrated DFT calculations. We then use DFT calculations to define and understand the changes in the NRVS spectra that arise from protonation and from opening the Fe–O–O angle. This study identifies four spectroscopic handles that will enable definition of the binding mode and protonation state of Fe^III-peroxy intermediates in mononuclear nonheme Fe enzymes. These structural differences are important in determining the frontier molecular orbitals available for reactivity

Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site (“H-cluster”) consists of a [4Fe–4S]_H cluster linked through a bridging cysteine to a [2Fe]_H subsite coordinated by CN^– and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fe_d). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe–H species fully consistent with widely accepted models of the catalytic cycle

Reaction Coordinate Leading to H₂ Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (H_hyd) in the [FeFe]-hydrogenases from both <i>Chlamydomonas reinhardtii</i> and <i>Desulfovibrio desulfuricans</i> using ⁵⁷Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe–H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe–H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that H_hyd is the catalytic state one step prior to H₂ formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H₂ bond formation by [FeFe]-hydrogenases

NRVS Studies of the Peroxide Shunt Intermediate in a Rieske Dioxygenase and Its Relation to the Native Fe^II O₂ Reaction

The Rieske dioxygenases are a major subclass of mononuclear nonheme iron enzymes that play an important role in bioremediation. Recently, a high-spin Fe^III–(hydro)­peroxy intermediate (BZDOp) has been trapped in the peroxide shunt reaction of benzoate 1,2-dioxygenase. Defining the structure of this intermediate is essential to understanding the reactivity of these enzymes. Nuclear resonance vibrational spectroscopy (NRVS) is a recently developed synchrotron technique that is ideal for obtaining vibrational, and thus structural, information on Fe sites, as it gives complete information on all vibrational normal modes containing Fe displacement. In this study, we present NRVS data on BZDOp and assign its structure using these data coupled to experimentally calibrated density functional theory calculations. From this NRVS structure, we define the mechanism for the peroxide shunt reaction. The relevance of the peroxide shunt to the native Fe^II/O₂ reaction is evaluated. For the native Fe^II/O₂ reaction, an Fe^III–superoxo intermediate is found to react directly with substrate. This process, while uphill thermodynamically, is found to be driven by the highly favorable thermodynamics of proton-coupled electron transfer with an electron provided by the Rieske [2Fe-2S] center at a later step in the reaction. These results offer important insight into the relative reactivities of Fe^III–superoxo and Fe^III–hydroperoxo species in nonheme Fe biochemistry

Synchrotron-based Nickel Mössbauer Spectroscopy

We used a novel experimental setup to conduct the first synchrotron-based ⁶¹Ni Mössbauer spectroscopy measurements in the energy domain on Ni coordination complexes and metalloproteins. A representative set of samples was chosen to demonstrate the potential of this approach. ⁶¹NiCr₂O₄ was examined as a case with strong Zeeman splittings. Simulations of the spectra yielded an internal magnetic field of 44.6 T, consistent with previous work by the traditional ⁶¹Ni Mössbauer approach with a radioactive source. A linear Ni amido complex, ⁶¹Ni­{N­(Si­Me₃)­Dipp}₂, where Dipp = C₆H₃-2,6-ⁱPr₂, was chosen as a sample with an “extreme” geometry and large quadrupole splitting. Finally, to demonstrate the feasibility of metalloprotein studies using synchrotron-based ⁶¹Ni Mössbauer spectroscopy, we examined the spectra of ⁶¹Ni-substituted rubredoxin in reduced and oxidized forms, along with [Et₄N]₂[⁶¹Ni­(SPh)₄] as a model compound. For each of the above samples, a reasonable spectrum could be obtained in ∼1 d. Given that there is still room for considerable improvement in experimental sensitivity, synchrotron-based ⁶¹Ni Mössbauer spectroscopy appears to be a promising alternative to measurements with radioactive sources

Geometric and Electronic Structure of the Mn(IV)Fe(III) Cofactor in Class Ic Ribonucleotide Reductase: Correlation to the Class Ia Binuclear Non-Heme Iron Enzyme

The class Ic ribonucleotide reductase (RNR) from <i>Chlamydia trachomatis</i> (<i>Ct</i>) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O₂. This reaction produces a stable Mn^IVFe^III cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the Mn^IVFe^III cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe^III, whereas MCD reflects the spin-allowed transitions mostly on the Mn^IV. We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn^IV at the site proximal to Phe₁₂₇. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d–d and oxo and OH^– to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn^IVFe^III cofactor as having a μ-oxo, μ-hydroxo core and a terminal hydroxo ligand on the Mn^IV. From DFT calculations, the Mn^IV at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH^– terminal ligand on this Mn^IV provides a high proton affinity that could gate radical translocation to the α (R1) subunit