Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Q_B Site of Bacterial Reaction Centers

Abstract

Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-10 molecules functioning as primary (QA) and secondary (QB) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the QB site semiquinone with nitrogens from the local protein environment in natural and 15N uniformly labeled reactions centers. 14N and 15N HYSCORE spectra of the QB semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from 14N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. 15N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen, and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (<0.1 MHz). These results clearly indicate that the QB semiquinone forms hydrogen bonds with two nitrogens, and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the QB site. Based on the quadrupole coupling constant, one nitrogen can only be assigned to Nδ of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen.

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2H and 15N Labeling, HYSCORE Spectroscopy, and DFT Modeling

“…Owing to the influence of the nuclear quadrupole interaction, no corresponding peak is detected near n I ( 14 N) in the 14 NH YSCORE spectrum shown in Figure2A. [16,17,19,43] To directly identify the origin of these signals, we constructed histidine and lysine auxotrophsf rom the E. coli JCB4023 strain. Inner membrane vesicles containing overproduced NarGHI werep urified and titrated to generate MSK D in equilibrium conditions.…”

“…Assigning the detected 1 Ha nd 14 Nn ucleit oaparticular chemical group is not straightforward. For this purpose, various strategies have been used, including (i)uniform enrichment of the protein in 2 H [12][13][14] or 15 N [15][16][17][18][19][20][21][22] or of the solvent thankst oH 2 O/ 2 H 2 Oe xchange experiments; [12,[23][24][25][26][27][28] (ii)theoretical calculations of hyperfine and quadrupolar parameters, [13,[29][30][31][32][33] (iii)reconstitution experimentsu sing exogenous quinones with various substituents or chemically labeled with 2 H, [29,34] and (iv) comparison with spectroscopic data obtained on model SQs measured in organic solvents. [35][36][37][38][39] Ad irect and more physiological strategy relies on in vivo isotope labeling of individual amino acids or sets of amino acids,w hich,h owever,r equires engineered auxotrophics trains supplemented with isotopically enriched amino acids.…”

“…Simulations are shown as red contour plots superimposedt othe experimentals pectrum.The spectrum (D) was simulated by considering two independent spin systems, each with asingle magnetically couplednitrogen nucleus. [19] Simulation parametersa nd their estimated errors are:(C) (A iso , T)( À158 mV is shown in Figure 3. It shows an intense unstructured isotropic radical signal centered at g av % 2.0045w ith a peak-to-peak line width of about 0.6 mT assigned to NarGHIbound MSK D with a Figure 4i nt he 0t o5MHz frequencyi nterval.…”

Colin A. Wraight, 1945–2014

“…He was not only brilliant and open in his thinking, but was a ''hands on'' person, and did experiments with us, turning and adjusting knobs, and sometimes even swearing. We collaborated on three topics, publishing four research papers: (1) With my PhD student Paul Jursinic, Colin and I showed, in 1978, a detailed and important relationship of membrane potential with microsecond delayed fluorescence from plants and algae, a topic he had earlier contributed to with Tony Crofts (see Jursinic et al 1978); (2) With several of our PhD students, Colin andI showed, in 1989 and1992, that bicarbonate, which my Lab had shown to be essential in the reduction and protonation of mobile quinone molecules in plants, algae and cyanobacteria (Shevela et al 2012), was not needed at all in a similar reaction in anoxygenic photosynthetic bacteria (Shopes et al 1989;Wang et al 1992); (3) With Vladimir Shinkarev, Colin's long-time brilliant associate, Colin and I succeeded, in 1997, in obtaining new information on the kinetics of oxygen evolution via chlorophyll a fluorescence measurements .…”

“…Early work identified the two-electron gate of Q B (Wraight 1977), and demonstrated once and for all that protonation was associated with electron transfer to this species (Wraight 1979a, b). This work continued throughout his career, probing the redox potential dependence of protonation (Maroti andWraight 1988a, b, 1997), crucial amino acids in the binding domain (Takahashi and Wraight 1990, Shinkarev et al 1993, Rinyu et al 2004, Martin et al 2010, Maroti et al 2015a and the effect of replacing the native ubiquinone with a variety of others (McComb et al 1990.…”