Probing the Iron−Substrate Orientation for Taurine/α-Ketoglutarate Dioxygenase Using Deuterium Electron Spin Echo Envelope Modulation Spectroscopy

Abstract

The structural relationship between substrate taurine and the non-heme Fe(II) center of taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD) was measured using electron spin echo envelope modulation (ESEEM) spectroscopy. Studies were conducted on TauD samples treated with NO, cosubstrate alphaKG, and either protonated or specifically deuterated taurine. Stimulated echo ESEEM data were divided to eliminate interference from 1H and 14N modulations and accentuate modulations from 2H. For taurine that was deuterated at the C1 position (adjacent to the sulfonate group), 2H ESEEM spectra show features that arise from dipole-dipole and deuterium nuclear quadrupole interactions from a single deuteron. Parallel measurements taken for taurine deuterated at both C1 and C2 show an additional ESEEM feature at the deuterium Larmor frequency. Analysis of these data at field positions ranging from g = 4 to g = 2 have allowed us to define the orientation of substrate taurine with respect to the magnetic axes of the Fe(II)-NO, S = 3/2, paramagnetic center. These results are discussed in terms of previous X-ray crystallographic studies and the proposed catalytic mechanism for this family of enzymes.

Insight into the mechanism of an iron dioxygenase by resolution of steps following the Fe IV ═O species

“…6) via direct interactions with a proton on the C 1 carbon as revealed by its substrate isotope sensitivity, similar to heme Fe IV ═O species (16)(17)(18). A short Fe─C 1 distance in TauD agrees with crystallographic data of the enzyme-substrate complex (28,29) and with electron spin-echo envelop modulation spectroscopy results that place a substrate C 1 deuteron 1.8 Å from the Fe II ─NO center (30). According to the generally described mechanism for this enzyme family (7,31,32), taurine oxidation proceeds via direct hydrogen atom transfer from C 1 of the substrate to the Fe IV ═O group yielding a Fe III ─OH and a substrate radical (Fig.…”

Experimental Correlation of Substrate Position with Reaction Outcome in the Aliphatic Halogenase, SyrB2

“…43–45 This complex is thought to be analogous to the putative {Fe-OO} 8 intermediate in the catalytic cycle, 43,46 and previous studies on taurine:2OG dioxygenase (TauD) have employed such complexes in combination with pulse EPR methods to determine the position of substrate C-H bonds. 40,41 In this work, we employed hyperfine sublevel correlation (HYSCORE) spectroscopy to obtain geometric information about the active site of SyrB2 by measuring hyperfine couplings to specifically 2 H-labeled Thr , Aba , and Nva substrates. The magnitude and orientation dependence of these hyperfine parameters provide spatial information about the labeled position relative to the metallocofactor; these data represent the first experimental structural data on the SyrB2:aminoacyl-SyrB1 complex and reveal the nature and extent of repositioning in the SyrB2 substrates exhibiting such widely divergent reactivities.…”

Modular behavior of tauD provides insight into the origin of specificity in α-ketoglutarate-dependent nonheme iron oxygenases

“…The orientation of the C-H bond with respect to the ferryl-oxo species is likely to be critical to the coupling of the ferryl-oxo to C-H cleavage. Spectroscopic studies have implied very short distances between the transferred hydrogen and the ferryl-oxo (22), and crystallographic and kinetic data for tauD indicate a wealth of interactions pertinent to substrate binding and reactivity (23)(24)(25). In particular, F159 stands out because of its relative position, with 4.25 Å separating C4 of F159 and the reactive C1 of taurine (Fig.…”