Rapid Freeze-Quench ⁵⁷Fe Mössbauer Spectroscopy:  Monitoring Changes of an Iron-Containing Active Site during a Biochemical Reaction

Abstract

Nuclear gamma resonance spectroscopy, also known as Mössbauer spectroscopy, is a technique that probes transitions between the nuclear ground state and a low-lying nuclear excited state. The nucleus most amenable to Mössbauer spectroscopy is 57Fe, and 57Fe Mössbauer spectroscopy provides detailed information about the chemical environment and electronic structure of iron. Iron is by far the most structurally and functionally diverse metal ion in biology, and 57Fe Mössbauer spectroscopy has played an important role in the elucidation of its biochemistry. In this article, we give a brief introduction to the technique and then focus on two recent exciting developments pertaining to the application of 57Fe Mössbauer spectroscopy in biochemistry. The first is the use of the rapid freeze-quench method in conjunction with Mössbauer spectroscopy to monitor changes at the Fe site during a biochemical reaction. This method has allowed for trapping and subsequent detailed spectroscopic characterization of reactive intermediates and thus has provided unique insight into the reaction mechanisms of Fe-containing enzymes. We outline the methodology using two examples: (1) oxygen activation by the non-heme diiron enzymes and (2) oxygen activation by taurine:alpha-ketoglutarate dioxygenase (TauD). The second development concerns the calculation of Mössbauer parameters using density functional theory (DFT) methods. By using the example of TauD, we show that comparison of experimental Mössbauer parameters with those obtained from calculations on model systems can be used to provide insight into the structure of a reaction intermediate.

Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase

“…Analysis of the spectra allows for determination of D and the x and y components of A͞g N ␤ N , as described in ref. 43. Theoretical simulations according to the spin-Hamiltonian formalism in the slow relaxation limit and the parameters given in Table 1 are shown as solid lines.…”

Evidence for C–H cleavage by an iron–superoxide complex in the glycol cleavage reaction catalyzed by myo -inositol oxygenase

“…Mössbauer and EPR spectra showed that treatment of recombinant Mus musculus MIOX isolated in its iron-free form from Escherichia coli with Fe(II) and O 2 leads to formation of an antiferromagnetically coupled diiron cluster in either the II͞III or III͞III oxidation state, depending on the O 2 ͞MIOX ratio and the presence or absence of a reductant (ascorbate or cysteine). Binding of MI was shown to perturb the spectra of both oxidation states in a manner consistent with direct coordination of the substrate to the cluster (5).All nonheme diiron oxygenases and oxidases characterized before MIOX activate O 2 with the II͞II oxidation state of the cofactor (11,12). For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated.…”

“…For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated. These complexes are generally proposed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave strong C-H or O-H bonds of their oxidation targets (8,(11)(12)(13)(14). Indeed, the diiron(III͞IV) cluster, X (15, 16), and the diiron(IV͞IV) cluster, Q (8, 13, 17), have been directly characterized in the R2 and soluble methane monooxygenase reactions, respectively.…”

“…All nonheme diiron oxygenases and oxidases characterized before MIOX activate O 2 with the II͞II oxidation state of the cofactor (11,12). For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated.…”

Bildung eines Oxoeisen(IV)‐Komplexes aus einem Nicht‐Häm‐Eisen(II)‐Komplex und H2O2 in wässriger Lösung bei pH 2–6

“…[1][2][3] Die intermediäre Bildung von Hydroxylradikalen [4] und/oder Eisen(IV)-Spezies [5] wurde auf der Basis von experimentellen Beobachtungen vorgeschlagen, und DFT-Rechnungen sprechen für das Auftreten hochvalenter Zwischenstufen. [6] Vor kurzem wurden Oxoeisen(IV)-Intermediate im Katalysezyklus von Taurin-a-ketoglutarat-Dioxygenase (TauD) identifiziert [7,8] und bei mehreren synthetischen Nicht-Häm-Eisenkomplexen charakterisiert, [9][10][11][12][13][14] wobei auch zwei hochaufgelöste Kristallstrukturanalysen vorgestellt wurden. [10,13] Anhand der spektroskopischen Merkmale dieser Präzedenz-fälle können nun auch ähnliche Zwischenstufen in anderen Systemen analysiert werden.…”