Orientation-Resolving Pulsed Electron Dipolar High-Field EPR Spectroscopy on Disordered Solids:  I. Structure of Spin-Correlated Radical Pairs in Bacterial Photosynthetic Reaction Centers

Abstract

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Transient EPR: using spin polarization in sequential radical pairs to study electron transfer in photosynthesis

“…From the angles obtained and a comparison with the X-ray structure, the authors conclude that the quinone undergoes a 60°rotation upon reduction. Examination of the angles obtained in Heinent et al (2007), however, shows that within experimental error the orientation of the dipolar coupling vector in the quinone g-tensor frame is the same as that given by the Xray structure and is also the same as that determined in Kandrashkin and van der Est (2001) and Savitsky et al (2007). Thus, the proposed 60°rotation of the quinone is about the dipolar coupling axis.…”

“…Calculation of the quasi-static K-band (van der Est et al 1993) and W-band (Prisner et al 1995; Kandrashkin and van der Est 2001) TREPR spectra using the geometry from the X-ray structure are in good agreement with the experimental spectra, which suggests that any light-induced reorientation is small. Recently, two independent studies (Heinent et al 2007;Savitsky et al 2007) have re-examined this issue and made use of additional data to refine the comparison. Both studies found evidence for the reorientation of Q A upon reduction, but the nature of the reorientation is significantly different in the two articles.…”

“…Both studies found evidence for the reorientation of Q A upon reduction, but the nature of the reorientation is significantly different in the two articles. In Savitsky et al (2007), an extensive analysis involving TREPR data as well as pulsed electron-electron double resonance (PELDOR) and relaxation-induced dipolar modulation enhancement (RIDME) data revealed a small but significant difference in the orientation of Q A derived from the EPR data compared to the X-ray structure. The slight difference suggested that the quinone undergoes a rotation of *14°about the carbonyl bonds upon reduction.…”

“…The spin polarization pattern as described by Eqs. 5 and 6 does not depend on this angle, which is referred to as the ''turning angle'' f QP in Savitsky et al (2007), and additional data such as dipolar spectra derived from PELDOR data are required to determine it. As discussed in Kandrashkin and van der Est (2001), the arbitrary scaling factor which is needed when TREPR data are fit also introduces an additional degree of freedom and prevents a unique solution of the geometry from being determined.…”

DEER Distance Measurements on Proteins

“…Orientation selection was analyzed for high-frequency DEER with enhanced symmetry of the relative orientation without (82) and with (83) conformational distribution of the paramagnetic center and for general symmetry for a cofactor pair in photosynthetic reaction centers (93). Analysis at X-band frequencies was desccribed for Cu 2+ -Cu 2+ (63, 94), Cu 2+ -nitroxide (63, 64) and iron-sulfur centre-nitroxide (94) pairs.…”