Probing Structural and Electronic Parameters in Randomly Oriented Metalloproteins by Orientation-Selective ENDOR Spectroscopy

g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance

“…While the g -tensor directions cannot be determined from one axial hyperfine tensor alone, constraints from multiple hyperfine tensors can substantially narrow the number of possible solutions. When combined with insight from previous g -tensor studies in similar systems, ,,− , two 13 C β(Cys) hyperfine tensors were found to be sufficient to model the g -tensor orientations in these cases.…”

“…In an orientation-selective pulsed EPR experiment, the hyperfine interactions of the paramagnetic center with the surrounding nuclei are measured as a function of the g -tensor orientation . This is achieved by taking measurements at different magnetic fields, thereby exciting only a subset of g values out of the full g -tensor at a time .…”

“…For biological iron–sulfur protein systems, the electronic structure and geometry of a cluster are primarily defined by the immediate ligand coordination environment, which can be further tuned by the pattern of hydrogen bonding with the backbone peptide matrix. − ,− All of these through-bond interactions can be addressed by spectroscopic approaches, such as pulsed electron and nuclear magnetic resonance (EPR and NMR, respectively) techniques. , EPR in particular is the method of choice for probing the physicochemical parameters that quantitatively define how the unpaired electron spin density delocalizes into the protein framework with respect to the molecular atomic coordinates. ,,− Currently, the most straightforward experimental approach for iron–sulfur protein systems with slow electronic relaxation rates is selective 15 N, 13 C, or 2 H isotope labeling of each amino acid type in the region local to the paramagnetic cluster-binding domain, regardless of whether pulsed EPR or NMR methods are employed. ,,, For this purpose, we have engineered a set of Escherichia coli amino acid auxotroph expression host strains for heterologous overproduction of selectively 15 N- and/or 13 C-amino acid isotope-labeled iron–sulfur proteins , and have applied these techniques to characterize the electronic structure of the reduced cluster site in the hyperthermophilic archaeal Rieske-type [2Fe-2S]­(His) 2 (Cys) 2 ferredoxin (ARF) from Sulfolobus solfataricus P1 (DSM 1616) − as a tractable model protein using two-dimensional, four-pulse electron spin–echo envelope modulation (ESEEM) [also called hyperfine sublevel correlation (HYSCORE)] spectroscopy. ,, …”

“…While modern pulsed EPR techniques in conjunction with selective amino acid isotope labeling can resolve and quantify hyperfine interactions between the unpaired electron spin and the magnetic nuclei of the input label, a deeper understanding of the enzymatic mechanism based on the precise interpretation of pulsed EPR data in the context of protein atomic coordinates greatly benefits from knowledge of the g -tensor orientation within the protein structural frame. , Some applications of the defined g -tensor directions to biological redox systems include the ability to analyze the separation distance and relative orientations between two paramagnetic centers by electron–electron dipolar interactions, − to assign dynamic changes in orientation of the paramagnetic center to possible conformational movements from enzyme catalytic action, − and, in more specialized cases, to locate possible proton positions relative to the paramagnetic center not readily available from moderate-resolution protein X-ray diffraction studies. − Most ideally, the g -tensor orientation of a protein-bound paramagnetic center would be determined precisely by single-crystal EPR . This direct approach is, however, often difficult to apply because of the strict requirement for large single crystals of proteins with appropriate molecular packing in the lattice.…”

“…This direct approach is, however, often difficult to apply because of the strict requirement for large single crystals of proteins with appropriate molecular packing in the lattice. In practice, orientation-selective pulsed EPR techniques, such as orientation-selective 1 H electron nuclear double resonance (ENDOR) spectroscopy, which probes through-space hyperfine interactions between the unpaired electron and the nuclei of the input label, can provide geometric information about the nuclei in the g -tensor reference frame. ,, When combined with the atomic coordinates of the target protein, this information is in certain cases sufficient for a full determination of the g -tensor directions of a paramagnetic center within the protein structural frame, to describe the mechanistic and functional aspects in the protein atomic coordinates using the high-resolution magnetic resonance data.…”