A crucial intermediate state in the reduction of QB to QBH2 involves the high energy intermediate QBH. state.(1) Direct observation of this state was achieved upon replacing the native ubiquinone with the low potential analog rhodoquinone (RQ) (2). Here we report the ENDOR spectrum of rhodosemiquinone at low and high pH measured in a mutant RC designed to reduce QB via B-branch electron transfer without interference from QA-. (3, 4). The peaks at 50.2 MHz and 51.7 MHz (dashed lines) are shifted from the proton Larmor frequency (∼53.2 MHz) and are assigned to hydrogen-bonded protons due to their line shape and position and their absence upon exchange in D2O (4). Upon shift to lower pH, these peaks diminish in amplitude and a new proton peak arises near 49.4 MHz (solid line). We attribute these changes to the protonation of rhodosemiquinone at lower pH. (Supported by NIH)(1) Graige et al (1996) J. Am. Chem Soc. 118, 9005-9016(2) Graige et al (1999) Biochemistry38, 11465-11473.(3) Paddock et al (2005) Biochemistry 44, 6920-6928.(4) Paddock et al (2009) Appl. Magn. Reson., in press.View Large Image | View Hi-Res Image | Download PowerPoint Slide