24 research outputs found
Компьютерные технологии проведения практических занятий по электротехнике
The paper gives the rationale behind the methodological approach of conducting practical training on electrical engineering. The approach features extensive guidance on students ' preliminary extra-curricular work in the process of preparation for the classes (drawing up schemes and graphs, chain calculation, etc.) The extra-curricular work is followed by the computer analysis of the chain along with the comparison of the result
Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins
Multiheme
proteins are important in energy conversion and biogeochemical
cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model
to elucidate roles of the interdomain interface on properties of iron
centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with
the full-length diheme c4 and its Met-to-His
ligand variants, were characterized by a variety of spectroscopic
and stability measurements. In both isolated domains, the heme iron
is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains,
and isolated c4-A is folded. In contrast,
the isolated c4-B is partially unfolded,
and the interface with c4-A guides folding
of this domain. The c4-A and c4-B domains have the propensity to interact even without
the polypeptide linker. Thermodynamic cycles have revealed properties
of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by
the interface. This study illustrates the effects of the interface
on tuning structural and redox properties of multiheme proteins and
enriches our understanding of redox-dependent complexation
The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (Fe<sub>B</sub>Mbs) Proceeds through a <i>trans</i>-Iron-Nitrosyl Dimer
Denitrifying NO reductases are transmembrane
protein complexes
that are evolutionarily related to heme/copper terminal oxidases.
They utilize a heme/nonheme diiron center to reduce two NO molecules
to N<sub>2</sub>O. Engineering a nonheme Fe<sub>B</sub> site within
the heme distal pocket of sperm whale myoglobin has offered well-defined
diiron clusters for the investigation of the mechanism of NO reduction
in these unique active sites. In this study, we use FTIR spectroscopy
to monitor the production of N<sub>2</sub>O in solution and to show
that the presence of a distal Fe<sub>B</sub><sup>II</sup> is not sufficient
to produce the expected product. However, the addition of a glutamate
side chain peripheral to the diiron site allows for 50% of a productive
single-turnover reaction. Unproductive reactions are characterized
by resonance Raman spectroscopy as dinitrosyl complexes, where one
NO molecule is bound to the heme iron to form a five-coordinate low-spin
{FeNO}<sup>7</sup> species with ν(FeNO)<sub>heme</sub> and ν(NO)<sub>heme</sub> at 522 and 1660 cm<sup>–1</sup>, and a second NO
molecule is bound to the nonheme Fe<sub>B</sub> site with a ν(NO)<sub>FeB</sub> at 1755 cm<sup>–1</sup>. Stopped-flow UV–vis
absorption coupled with rapid-freeze-quench resonance Raman spectroscopy
provide a detailed map of the reaction coordinates leading to the
unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to Fe<sub>B</sub> is kinetically favored and occurs prior to the binding of
a second NO to the heme iron, leading to a (six-coordinate low-spin
heme-nitrosyl/Fe<sub>B</sub>-nitrosyl) transient dinitrosyl complex
with characteristic ν(FeNO)<sub>heme</sub> at 570 ± 2 cm<sup>–1</sup> and ν(NO)<sub>FeB</sub> at 1755 cm<sup>–1</sup>. Without the addition of a peripheral glutamate, the dinitrosyl
complex is converted to a dead-end product after the dissociation
of the proximal histidine of the heme iron, but the added peripheral
glutamate side chain in Fe<sub>B</sub>Mb2 lowers the rate of dissociation
of the promixal histidine which in turn allows the (six-coordinate
low-spin heme-nitrosyl/Fe<sub>B</sub>-nitrosyl) transient dinitrosyl
complex to decay with production of N<sub>2</sub>O at a rate of 0.7
s<sup>–1</sup> at 4 °C. Taken together, our results support
the proposed trans mechanism of NO reduction in NORs
Vibrational Analysis of Mononitrosyl Complexes in Hemerythrin and Flavodiiron Proteins: Relevance to Detoxifying NO Reductase
Flavodiiron proteins (FDPs) play important roles in the
microbial nitrosative stress response in low-oxygen environments by
reductively scavenging nitric oxide (NO). Recently, we showed that
FMN-free diferrous FDP from <i>Thermotoga maritima</i> exposed
to 1 equiv NO forms a stable diiron-mononitrosyl complex (deflavo-FDP(NO))
that can react further with NO to form N<sub>2</sub>O [Hayashi, T.; Caranto, J. D.; Wampler, D. A; Kurtz, D. M., Jr.; Moënne-Loccoz, P. Biochemistry 2010, 49, 7040−7049]. Here we report resonance Raman and low-temperature
photolysis FTIR data that better define the structure of this diiron-mononitrosyl
complex. We first validate this approach using the stable diiron-mononitrosyl
complex of hemerythrin, Hr(NO), for which we observe a ν(NO)
at 1658 cm<sup>–1</sup>, the lowest ν(NO) ever reported
for a nonheme {FeNO}<sup>7</sup> species. Both deflavo-FDP(NO) and
the mononitrosyl adduct of the flavinated FPD (FDP(NO)) show ν(NO)
at 1681 cm<sup>–1</sup>, which is also unusually low. These
results indicate that, in Hr(NO) and FDP(NO), the coordinated NO is
exceptionally electron rich, more closely approaching the Fe(III)(NO<sup>–</sup>) resonance structure. In the case of Hr(NO), this
polarization may be promoted by steric enforcement of an unusually
small FeNO angle, while in FDP(NO), the Fe(III)(NO<sup>–</sup>) structure may be due to a semibridging electrostatic interaction
with the second Fe(II) ion. In Hr(NO), accessibility and steric constraints
prevent further reaction of the diiron-mononitrosyl complex with NO,
whereas in FDP(NO) the increased nucleophilicity of the nitrosyl group
may promote attack by a second NO to produce N<sub>2</sub>O. This
latter scenario is supported by theoretical modeling [Blomberg, L. M.; Blomberg, M. R.; Siegbahn, P. E. J. Biol.
Inorg. Chem. 2007, 12, 79−89]. Published vibrational
data on bioengineered models of denitrifying heme-nonheme NO reductases
[Hayashi, T.; Miner, K. D.; Yeung, N.; Lin, Y.-W.; Lu, Y.; Moënne-Loccoz, P. Biochemistry 2011, 50, 5939−5947] support a similar mode of activation of a heme {FeNO}<sup>7</sup> species by the nearby nonheme Fe(II)
A Nonheme, High-Spin {FeNO}<sup>8</sup> Complex that Spontaneously Generates N<sub>2</sub>O
One-electron reduction of [Fe(NO)-(N3PyS)]BF<sub>4</sub> (<b>1</b>) leads to the production of the metastable
nonheme {FeNO}<sup>8</sup> complex, [Fe(NO)(N3PyS)] (<b>3</b>). Complex <b>3</b> is a rare example of a high-spin (<i>S</i> = 1)
{FeNO}<sup>8</sup> and is the first example, to our knowledge, of
a mononuclear nonheme {FeNO}<sup>8</sup> species that generates N<sub>2</sub>O. A second, novel route to <b>3</b> involves addition
of Piloty’s acid, an HNO donor, to an Fe<sup>II</sup> precursor.
This work provides possible new insights regarding the mechanism of
nitric oxide reductases
The Hemophore HasA from <i>Yersinia pestis</i> (HasA<sub>yp</sub>) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change
Hemophores from <i>Serratia
marcescens</i> (HasA<sub>sm</sub>) and <i>Pseudomonas aeruginosa</i> (HasA<sub>p</sub>) bind hemin between two loops, which harbor the
axial ligands H32
and Y75. Hemin binding to the Y75 loop triggers closing of the H32
loop and enables binding of H32. Because <i>Yersinia pestis</i> HasA (HasA<sub>yp</sub>) presents a Gln at position 32, we determined
the structures of apo- and holo-HasA<sub>yp</sub>. Surprisingly, the
Q32 loop in apo-HasA<sub>yp</sub> is already in the closed conformation,
but no residue from the Q32 loop binds hemin in holo-HasA<sub>yp</sub>. In agreement with the minimal reorganization between the apo- and
holo-structures, the hemin on-rate is too fast to detect by conventional
stopped-flow measurements
A Nonheme Iron(III) Superoxide Complex Leads to Sulfur Oxygenation
A new alkylthiolate-ligated nonheme iron complex, FeII(BNPAMe2S)Br (1), is reported. Reaction
of 1 with O2 at −40 °C, or reaction
of
the ferric form with O2•– at −80
°C, gives a rare iron(III)-superoxide intermediate, [FeIII(O2)(BNPAMe2S)]+ (2), characterized by UV–vis, 57Fe Mössbauer,
ATR-FTIR, EPR, and CSIMS. Metastable 2 then converts
to an S-oxygenated FeII(sulfinate) product via a sequential
O atom transfer mechanism involving an iron-sulfenate intermediate.
These results provide evidence for the feasibility of proposed intermediates
in thiol dioxygenases