Site-specific oxidation of the [Fe4S4] cubanes in high-potential iron sulfur proteins as probed by EPR and orientation-selective proton ENDOR spectroscopy:Ectothiorhodospira halophila I versusRhodocyclus tenuis

Abstract

Electron paramagnetic resonance (EPR) and proton ENDOR (electron-nuclear double resonanee) spectroscopies were used to analyze the struetural and eleetronic parameters of the oxidized [Fe4S4] cubane clusters in high-potential iron sulfur proteins (HiPIPs) from Ectothiorhodospira halophila (HiPIP I) and Rhodocyclus tenuis. The E. halophila HiPIP I EPR spectra at X-and Q-band revealed a dominant species (simulated with gm~ = 2.1425, g,n, = 2.0315, gmm = 2.0296) anda minor speeies (ea. 5-10% contribution) which was not analyzed further. For R. tenuis HiPIP the EPR spectrum contained a single species only (g~x = 2.1140, g~,, = 2.0392, gm~, = 2.0215), i.e., with gmax significantly smaller than that of the E. halophila protein. Orientation-selected proton ENDOR spectra of HiPIP I of E. halophila were reconstructed by simulation with slight modifications of the crystal structure data. ENDOR from two mutants, F36S and F36G, of E. halophila HiPIP I gave evidente fora common assignment of a HB2 proton of a phenylalanine residue (36 and 44, respectively, in isoenzymes I and II as reported earlier) interacting with the mixed-valence pair iron ions Fe2 and Fe3. For R. tenuis HiPIE the ENDOR spectra were assigned to arise from Fe3 and Fe4 as mixedvalence pair under the assumption of ah unchanged intrinsic g-tensor symmetry. The resulting site specificity of the cubane oxidation was discussed in relation to structural requirements and redox potentials of the two HiPIPs.