11 research outputs found
A New Paradigm for Electron Transfer through <i>Escherichia coli</i> Nitrate Reductase A
We have investigated the role of
redox cooperativity in defining
the functional relationship among the three membrane-associated prosthetic
groups of <i>Escherichia coli</i> nitrate reductase A: the
two hemes (<i>b</i><sub>D</sub> and <i>b</i><sub>P</sub>) of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster
(FS4) of the electron-transfer subunit (NarH). Previously published
analyses of potentiometric titrations have exhibited the following
anomalous behaviors: (i) fits of titration data for heme <i>b</i><sub>p</sub> and the [3Fe-4S] cluster exhibited two apparent components;
(ii) heme <i>b</i><sub>D</sub> titrated with an apparent
electron stoichiometry (<i>n</i>) of <1.0; and (iii)
the binding of quinol oxidation inhibitors shifted the reduction potentials
of both hemes despite there being only a single quinol oxidation site
(Q-site) in close juxtaposition with heme <i>b</i><sub>D</sub>. Furthermore, both hemes appeared to be affected despite the absence
of major structural shifts upon inhibitor binding, as judged by X-ray
crystallography, or evidence of a second Q-site in the vicinity of
heme <i>b</i><sub>P</sub>. In a re-examination of the redox
behavior of hemes <i>b</i><sub>D</sub> and <i>b</i><sub>P</sub> and FS4, we have developed a cooperative redox model
of cofactor interaction. We show that anticooperative interactions
provide an explanation for the anomalous behavior. We propose that
the role of such anticooperative redox behavior <i>in vivo</i> is to facilitate transmembrane electron transfer across an energy-conserving
membrane against an electrochemical potential
Q‑Site Occupancy Defines Heme Heterogeneity in <i>Escherichia coli</i> Nitrate Reductase A (NarGHI)
The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains
two <i>b</i>-type
hemes, both of which are the highly anisotropic low-spin type. Heme <i>b</i><sub>D</sub> is distal to NarGH and constitutes part of
the quinone binding and oxidation site (Q-site) through the axially
coordinating histidine-66 residue and one of the heme <i>b</i><sub>D</sub> propionate groups. Bound quinone participates in hydrogen
bonds with both the imidazole of His66 and the heme propionate, rendering
the EPR spectrum of the heme <i>b</i><sub>D</sub> sensitive
to Q-site occupancy. As such, we hypothesize that the heterogeneity
in the heme <i>b</i><sub>D</sub> EPR signal arises from
the differential occupancy of the Q-site. In agreement with this,
the heterogeneity is dependent upon growth conditions but is still
apparent when NarGHI is expressed in a strain lacking cardiolipin.
Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A
and NarI-K86A, and is collapsible by the binding of inhibitors. We
found that the two main <i>g</i><sub><i>z</i></sub> components of heme <i>b</i><sub>D</sub> exhibit differences
in reduction potential and pH dependence, which we posit is due to
differential Q-site occupancy. Specifically, in a quinone-bound state,
heme <i>b</i><sub>D</sub> exhibits an <i>E</i><sub>m,8</sub> of −35 mV and a pH dependence of −40
mV pH<sup>–1</sup>. In the quinone-free state, however, heme <i>b</i><sub>D</sub> titrates with an <i>E</i><sub>m,8</sub> of +25 mV and a pH dependence of −59 mV pH<sup>–1</sup>. We hypothesize that quinone binding modulates the electrochemical
properties of heme <i>b</i><sub>D</sub> as well as its EPR
properties
Comparative Proteomic and Metabolomic Analysis of Staphylococcus warneri SG1 Cultured in the Presence and Absence of Butanol
The complete genome of the solvent
tolerant Staphylococcus
warneri SG1 was recently published. This Gram-positive
bacterium is tolerant to a large spectrum of organic solvents including
short-chain alcohols, alkanes, esters and cyclic aromatic compounds.
In this study, we applied a two-dimensional liquid chromatography
(2D-LC) mass spectrometry (MS) shotgun approach, in combination with
quantitative 2-MEGA (dimethylation after guanidination) isotopic labeling,
to compare the proteomes of SG1 grown under butanol-free and butanol-challenged
conditions. In total, 1585 unique proteins (representing 65% of the
predicted open reading frames) were identified, covering all major
metabolic pathways. Of the 967 quantifiable proteins by 2-MEGA labeling,
260 were differentially expressed by at least 1.5-fold. These proteins
are involved in energy metabolism, oxidative stress response, lipid
and cell envelope biogenesis, or have chaperone functions. We also
applied differential isotope labeling LC-MS to probe metabolite changes
in key metabolic pathways upon butanol stress. This is the first comprehensive
proteomic and metabolomic study of S. warneri SG1 and presents an important step toward understanding its physiology
and mechanism of solvent tolerance
Comparative Proteomic and Metabolomic Analysis of Staphylococcus warneri SG1 Cultured in the Presence and Absence of Butanol
The complete genome of the solvent
tolerant Staphylococcus
warneri SG1 was recently published. This Gram-positive
bacterium is tolerant to a large spectrum of organic solvents including
short-chain alcohols, alkanes, esters and cyclic aromatic compounds.
In this study, we applied a two-dimensional liquid chromatography
(2D-LC) mass spectrometry (MS) shotgun approach, in combination with
quantitative 2-MEGA (dimethylation after guanidination) isotopic labeling,
to compare the proteomes of SG1 grown under butanol-free and butanol-challenged
conditions. In total, 1585 unique proteins (representing 65% of the
predicted open reading frames) were identified, covering all major
metabolic pathways. Of the 967 quantifiable proteins by 2-MEGA labeling,
260 were differentially expressed by at least 1.5-fold. These proteins
are involved in energy metabolism, oxidative stress response, lipid
and cell envelope biogenesis, or have chaperone functions. We also
applied differential isotope labeling LC-MS to probe metabolite changes
in key metabolic pathways upon butanol stress. This is the first comprehensive
proteomic and metabolomic study of S. warneri SG1 and presents an important step toward understanding its physiology
and mechanism of solvent tolerance
Interactions between the heme face and surrounding residues.
<p>Mutations to the residues depicted are able to affect the planarity of the porphyrin ring. Pyrrole rings are denoted as (A) top, distal (B) top, proximal (C) bottom, proximal (D) bottom, distal. (pdb: 2WDV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032641#pone.0032641-Ruprecht1" target="_blank">[31]</a>).</p
The hydrogen-bonding network around the heme propionates.
<p>The distal propionate is highlighted on the left while the proximal propionate is shown on the right. Each propionate is held in position by residues on either side. Mutation of SdhD<sup>R20</sup> and SdhC<sup>R31</sup> to a Leu results in the propionate shifting towards the other “girder” (light arrows). Mutation of SdhC<sup>H91</sup> and SdhD<sup>Q78</sup> to a Leu causes a shift of the propionate in the other direction (dark arrows). Note the twisting of the porphyrin ring into a saddled conformation in this structure. (pdb: 2WDV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032641#pone.0032641-Ruprecht1" target="_blank">[31]</a>).</p
Residues selected for mutation.
<p>Upon mutation, residues that result in a shift towards the “sharp” EPR spectrum are colored in yellow while positions causing a shift towards the “broad” EPR spectrum are colored in blue. Residues that did not affect the EPR spectrum when mutated are shown in green. (pdb: 1NEK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032641#pone.0032641-Yankovskaya1" target="_blank">[4]</a>).</p
Redox characteristics, physiological and non-physiological activity, and ROS assays of wild-type and Sdh variants.
<p>Turnover numbers have all been normalized to FAD content. Activity data were averaged from at least three independent measurements.</p
Comparative Proteomic and Metabolomic Analysis of Staphylococcus warneri SG1 Cultured in the Presence and Absence of Butanol
The complete genome of the solvent
tolerant Staphylococcus
warneri SG1 was recently published. This Gram-positive
bacterium is tolerant to a large spectrum of organic solvents including
short-chain alcohols, alkanes, esters and cyclic aromatic compounds.
In this study, we applied a two-dimensional liquid chromatography
(2D-LC) mass spectrometry (MS) shotgun approach, in combination with
quantitative 2-MEGA (dimethylation after guanidination) isotopic labeling,
to compare the proteomes of SG1 grown under butanol-free and butanol-challenged
conditions. In total, 1585 unique proteins (representing 65% of the
predicted open reading frames) were identified, covering all major
metabolic pathways. Of the 967 quantifiable proteins by 2-MEGA labeling,
260 were differentially expressed by at least 1.5-fold. These proteins
are involved in energy metabolism, oxidative stress response, lipid
and cell envelope biogenesis, or have chaperone functions. We also
applied differential isotope labeling LC-MS to probe metabolite changes
in key metabolic pathways upon butanol stress. This is the first comprehensive
proteomic and metabolomic study of S. warneri SG1 and presents an important step toward understanding its physiology
and mechanism of solvent tolerance
Ferric heme EPR lineshapes of the two heme conformations.
<p>The signal in wild-type Sdh is a mixture of a sharp and broad species, exemplified in the SdhC<sup>H30A</sup> and SdhC<sup>T37I</sup> variants, respectively.</p