34 research outputs found
Two-dimensional pulsed electron spin resonance characterization of 15N-labeled archaeal Rieske-type ferredoxin
AbstractTwo-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly 15N-labeled archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems
Eseem And Hyscore Analysis Of QA- In Native And 15N Labeled Reaction Centers From Rhodobacter sphaeroides
Eseem And Hyscore Analysis Of QA- In Native And 15N Labeled Reaction Centers From Rhodobacter sphaeroides
Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis
Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa(3)-600 Menaquinol Oxidase from Bacillus subtilis
Exploring by pulsed EPR the electronic structure of ubisemiquinone bound at the QH site of cytochrome bo3 from Escherichia coli with in vivo 13C-labeled methyl and methoxy substituents
The cytochrome bo(3) ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O(2) to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo(3) as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 (13)C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group (13)C-labeled. The (13)C-labeled quinone isolated from cytochrome bo(3) was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity
Hydrogen bonding and spin density distribution in the Q B semiquinone of bacterial reaction centers and comparison with the Q A site
In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (Q(A)) and secondary (Q(B)) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the Q(A) and Q(B) sites, using samples of (15)N-labeled reaction centers, with the native high spin Fe(2+) exchanged for diamagnetic Zn(2+), prepared in (1)H(2)O and (2)H(2)O solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the Q(A) SQ by Flores et al. (Biophys. J. 2007, 92, 671–682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6–5.4 MHz. HYSCORE was then applied to the Q(B) SQ where we found proton lines corresponding to T~5.2, 3.7 MHz and T~1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/MM) calculations, employing a model of the Q(B) site, were used to assign the observed couplings to specific hydrogen bonding interactions with the Q(B) SQ. These calculations allow us to assign the T=5.2 MHz proton to the His-L190 N(δ)H…O(4) (carbonyl) hydrogen bonding interaction. The T =3.7 MHz spectral feature most likely results from hydrogen bonding interactions of O1 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the Q(B) site show less asymmetric distribution of unpaired spin density over the SQ than seen for the Q(A) site, consistent with available experimental data for (13)C and (17)O carbonyl hyperfine couplings. The implications of these interactions for Q(B) function and comparisons with the Q(A) site are discussed
Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome <i>aa</i><sub>3</sub>‑600 Menaquinol Oxidase from <i>Bacillus subtilis</i>
Cytochrome <i>aa</i><sub>3</sub>-600 is a terminal oxidase
in the electron transport pathway that contributes to the electrochemical
membrane potential by actively pumping protons. A notable feature
of this enzyme complex is that it uses menaquinol as its electron
donor instead of cytochrome <i>c</i> when it reduces dioxygen
to water. The enzyme stabilizes a menasemiquinone radical (SQ) at
a high affinity site that is important for catalysis. One of the residues
that interacts with the semiquinone is Arg70. We have made the R70H
mutant and have characterized the menasemiquinone radical by advanced
X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong
isotropic hyperfine coupling (<i>a</i><sub><sup>14</sup>N</sub> ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen
originates from a histidine side chain, based on its quadrupole coupling
constant, <i>e</i><sup>2</sup><i>qQ</i>/<i>h</i> = 1.44 MHz, typical for protonated imidazole nitrogens.
In the wild-type cyt <i>aa</i><sub>3</sub>-600, the SQ is
instead hydrogen bonded with N<sub>ε</sub> from the Arg70 side
chain. Analysis of the <sup>1</sup>H 2D electron spin echo envelope
modulation (ESEEM) spectra shows that the mutation also changes the
number and strength of the hydrogen bonds between the SQ and the surrounding
protein. Despite the alterations in the immediate environment of the
SQ, the R70H mutant remains catalytically active. These findings are
in contrast to the equivalent mutation in the close homologue, cytochrome <i>bo</i><sub>3</sub> ubiquinol oxidase from <i>Escherichia
coli</i>, where the R71H mutation eliminates function