EPR, ENDOR, and Special TRIPLE measurements of P•+ in wild type and modified reaction centers from Rb. sphaeroides

Abstract

The influence of the protein environment on the primary electron donor, P, a bacteriochlorophyll a dimer, of reaction centers from Rhodobacter sphaeroides, has been investigated using electron paramagnetic resonance and electron nuclear double resonance spectroscopy. These techniques were used to probe the effects on P that are due to alteration of three amino acid residues, His L168, Asn L170, and Asn M199. The introduction of Glu at L168, Asp at L170, or Asp at M199 changes the oxidation/ reduction midpoint potential of P in a pH-dependent manner (Williams et al. (2001) Biochemistry 40, 15403-15407). For the double mutant His L168 to Glu and Asn at L170 to Asp, excitation results in electron transfer along the A-side branch of cofactors at pH 7.2, but at pH 9.5, a long-lived state involving B-side cofactors is produced (Haffa et al. (2004) J Phys Chem B 108, 4-7). Using electron paramagnetic resonance spectroscopy, the mutants with alterations of each of the three individual residues and a double mutant, with changes at L168 and L170, were found to have increased linewidths of 10.1-11.0 G compared to the linewidth of 9.6 G for wild type. The Special TRIPLE spectra were pH dependent, and at pH 8, the introduction of aspartate at L170 increased the spin density ratio, q L /q M , to 6.1 while an aspartate at the symmetry related position, M199, decreased the ratio to 0.7 compared to the value of 2.1 for wild type. These results indicate that the energy of the two halves of P changes by about 100 meV due to the mutations and are consistent with the interpretation that electrostatic interactions involving these amino acid residues contribute to the switch in pathway of electron transfer.

Spin densities from subsystem density-functional theory: Assessment and application to a photosynthetic reaction center complex model

“…In spite of the fact that the cofactors in the PRC are organized in an almost two-fold symmetry around the SP, the electron transfer proceeds only along one of the two branches. [3][4][5] According to previous theoretical 6- 10 and experimental studies [11][12][13][14] the reason for this unidirectionality could be related to a spin-density asymmetry already in SP • + , which might be induced by the a) Electronic mail: m.pavanello@chem.leidenuniv.nl. b) Electronic mail: j.neugebauer@tu-braunschweig.…”

Introduction to magnetic resonance methods in photosynthesis

“…Obviously, similar effects play a role in the more extended π-electron systems of photosynthetic cofactors. Detailed investigations of the distribution of spin density (Allen et al 2009 ) and G- tensor of these cofactors reveal subtle differences in hydrogen bonding and conformations. The response of the extended π-electron systems of these cofactors to the protein environment seems to be one of the mechanisms by which the protein can fine tune the electronic properties of the cofactors to function optimally.…”

Comparison of bacterial reaction centers and photosystem II

“…In addition to hydrogen bonds, preferential changes in the energetics of P were made by the alteration of the electrostatic interactions between P and the surrounding protein by either introducing or removing ionizable amino acid residues (Williams et al 2001;Johnson and Parson 2002;Allen et al 2008). For example, introducing a negative charge at M199 resulted in a large shift with only 41% of the spin being on P L , while introducing a negative charge at the symmetry-related position increased the relative spin density to 86% compared to 68% for wild type.…”