Interactions of Intermediate Semiquinone with Surrounding Protein Residues at the Q_H Site of Wild-Type and D75H Mutant Cytochrome bo₃ from Escherichia coli

Abstract

Selective 15N isotope labeling of the cytochrome bo3 ubiquinol oxidase from E. coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from R71, H98, and Q101 residues and peptide nitrogens from R71 and H98 residues around the semiquinone (SQ) at the high-affinity QH site. The 2D ESEEM (HYSCORE) data have directly identified the Nε of R71 as an H-bond donor carrying the largest amount of the unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the QH site and strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that the Nε from a histidine residue, presumably H75, carries most of the unpaired spin density instead of the Nε of R71, as in the wild-type bo3. However, the detailed characterization of the weakly coupled 15Ns from selective labeling of R71 and Q101 in D75H was precluded by overlap of the 15N lines with the much stronger ~1.6 MHz line from quadrupole triplet of the strongly coupled 14Nε from H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied in order to probe the contribution of R71 and Q101 to the 15N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory based calculations to model the available information about 1H, 15N and 13C hyperfine couplings for the QH site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone’s electron transfer function.

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

“…[10,11,29,66] This observation has been discussed with regard to the quinone electron transfer role as one-electron (Q A ,A 1 , and Q H )o rt wo-electron transfer agent (Q B ,q uinonesi nv itro) coupledt ot he binding or releaseo ft wo protons. [44] Whereas the number of studied systemsi st oo low for substantial conclusions, the present study providesa nu nprecedented example of aq uinone with an extremelyh igh asymmetrics pin density distribution for the MSK D intermediate whereas menaquinols in the Q D site are in dynamic equilibrium with the quinone pool and bind to release two electrons and two protons before unbinding in the oxidized menaquinone form. It can therefore be concluded that the production within aQsite of ah ighly asymmetrics pin density distribution for the SQ intermediate does not systematically translate into ar ole as ao neelectron transfer agent forthis Qs ite.…”

“…[40][41][42] With this strategy,s electively 15 N-labeled cyt bo 3 samples from E. coli were used in 15 NH YSCORE experiments to identify the amino acids interacting with the stabilized ubisemiquinone species bounda tt he high affinity quinone binding site (Q H )o ft he wild-type enzymeo ri naQ-site mutant. [43,44] Ar everse labelinga pproachi nw hich this enzyme was uniformly 15 N-labeled except for selected amino acid types has also been used. [44] In this work, 2 Ha nd 15 Ni nvivo selective labelingi su sed to resolve quinone-protein interactions in the respiratory nitrate reductase (Nar) from E. coli,awidely distributed prokaryotic enzyme that allows anaerobic respiration with nitrate as at erminal electron acceptor.T his membrane-bound heterotrimeric complex( NarGHI) couplest he oxidation of membrane quinols at ap eriplasmically oriented Qs ite to the cytoplasmic twoelectron reduction of nitrate into nitrite.…”

“…[43,44] Ar everse labelinga pproachi nw hich this enzyme was uniformly 15 N-labeled except for selected amino acid types has also been used. [44] In this work, 2 Ha nd 15 Ni nvivo selective labelingi su sed to resolve quinone-protein interactions in the respiratory nitrate reductase (Nar) from E. coli,awidely distributed prokaryotic enzyme that allows anaerobic respiration with nitrate as at erminal electron acceptor.T his membrane-bound heterotrimeric complex( NarGHI) couplest he oxidation of membrane quinols at ap eriplasmically oriented Qs ite to the cytoplasmic twoelectron reduction of nitrate into nitrite. [47,48] NarGHI turnover inducesan et translocation of protons across the membrane, which contributes to maintaining the transmembrane proton gradientt hat drives, for instance, ATPs ynthesis.…”

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochromebo3fromEscherichia coli

“…It is known that the strongly H-bonded protons H1 and H2 are on the O 1 side of the SQ H , however, their assignment to residues D75 and R71 is ambiguous. 18,20 Parallel MD runs were set up with proton H1 being assigned either to the β-carboxyl of D75 or to the N ε of R71 to test both possibilities.…”

“…25 However, the DFT calculations utilizing different simplified models of either the ubiquinone anion-radical or neutral radical H-bonding failed to reproduce all experimentally determined 1 H (H-bonds, 5′-methyl), 13 C (carbonyls), and 14 N (nitrogen H-bond donors) couplings simultaneously. 20,28–30 Hence, the SQ H protonation state, as well as the general hydrogen bond network, remain uncertain. What is required to clarify the situation is a more precise, experimentally characterized model of the SQ H binding in cyt bo 3 including the orientations of the H-bonds and the corresponding donor for each strongly coupled proton.…”