Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy.

Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300 kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids.

Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency.