11 research outputs found
Nitrogen Fixation via a Terminal Fe(IV) Nitride
Terminal iron nitrides (Feī¼N)
have been proposed as intermediates
of (bio)Ācatalytic nitrogen fixation, yet experimental evidence to
support this hypothesis has been lacking. In particular, no prior
synthetic examples of terminal Feī¼N species have been derived
from N<sub>2</sub>. Here we show that a nitrogen-fixing FeāN<sub>2</sub> catalyst can be protonated to form a neutral FeĀ(NNH<sub>2</sub>) hydrazido(2ā) intermediate, which, upon further protonation,
heterolytically cleaves the NāN bond to release [Fe<sup>IV</sup>ī¼N]<sup>+</sup> and NH<sub>3</sub>. These observations provide
direct evidence for the viability of a Chatt-type (distal) mechanism
for Fe-mediated N<sub>2</sub>-to-NH<sub>3</sub> conversion. The physical
oxidation state range of the Fe complexes in this transformation is
buffered by covalency with the ligand, a feature of possible relevance
to catalyst design in synthetic and natural systems that facilitate
multiproton/multielectron redox processes
Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase PāCluster
Nitrogenase catalyzes
the multielectron reduction of dinitrogen
to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds
through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco).
In the reduced, all-ferrous (PN) state, the P-cluster is
coordinated by six cysteine residues. Upon two-electron oxidation
to the P2+ state, the P-cluster undergoes conformational
changes in which a highly conserved oxygen-based residue (a Ser or
a Tyr) and a backbone amide additionally ligate the cluster. Previous
studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, Ī²Ser188,
was mutated to a noncoordinating residue, Ala, the P-cluster became
redox-labile and reversibly lost two of its eight Fe centers. Surprisingly,
the Av strain with a MoFeP variant that lacked the
serine ligand (Av Ī²Ser188Ala MoFeP) displayed
the same diazotrophic growth and in vitro enzyme
turnover rates as wild-type Av MoFeP, calling into
question the necessity of this conserved ligand for nitrogenase function.
Based on these observations, we hypothesized that Ī²Ser188 plays
a role in protecting the P-cluster under nonideal conditions. Here,
we investigated the protective role of Ī²Ser188 both in vivo and in vitro by characterizing
the ability of Av Ī²Ser188Ala cells to grow
under suboptimal conditions (high oxidative stress or Fe limitation)
and by determining the tendency of Ī²Ser188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that Ī²Ser188 (1)
increases Av cell survival upon exposure to oxidative
stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and
(3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of
nitrogenase to protect the P-cluster via Ser ligation, which is a
previously unidentified functional role of the Ser residue in redox
proteins and adds to the expanding functional roles of non-Cys ligands
to FeS clusters
Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex
The
rebound mechanism for alkane hydroxylation was invoked over
40 years ago to help explain reactivity patterns in cytochrome P450,
and subsequently has been used to provide insight into a range of
biological and synthetic systems. Efforts to model the rebound reaction
in a synthetic system have been unsuccessful, in part because of the
challenge in preparing a suitable metal-hydroxide complex at the correct
oxidation level. Herein we report the synthesis of such a complex.
The reaction of this species with a series of substituted radicals
allows for the direct interrogation of the rebound process, providing
insight into this uniformly invoked, but previously unobserved process
Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex
The
rebound mechanism for alkane hydroxylation was invoked over
40 years ago to help explain reactivity patterns in cytochrome P450,
and subsequently has been used to provide insight into a range of
biological and synthetic systems. Efforts to model the rebound reaction
in a synthetic system have been unsuccessful, in part because of the
challenge in preparing a suitable metal-hydroxide complex at the correct
oxidation level. Herein we report the synthesis of such a complex.
The reaction of this species with a series of substituted radicals
allows for the direct interrogation of the rebound process, providing
insight into this uniformly invoked, but previously unobserved process
Setting an Upper Limit on the Myoglobin Iron(IV)Hydroxide p<i>K</i><sub>a</sub>: Insight into Axial Ligand Tuning in Heme Protein Catalysis
To provide insight into the ironĀ(IV)Āhydroxide
p<i>K</i><sub>a</sub> of histidine ligated heme proteins,
we have probed the
active site of myoglobin compound II over the pH range of 3.9ā9.5,
using EXAFS, MoĢssbauer, and resonance Raman spectroscopies.
We find no indication of ferryl protonation over this pH range, allowing
us to set an upper limit of 2.7 on the ironĀ(IV)Āhydroxide p<i>K</i><sub>a</sub> in myoglobin. Together with the recent determination
of an ironĀ(IV)Āhydroxide p<i>K</i><sub>a</sub> ā¼ 12
in the thiolate-ligated heme enzyme cytochrome P450, this result provides
insight into Natureās ability to tune catalytic function through
its choice of axial ligand
Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme
We
report on the protonation state of <i>Helicobacter pylori</i> catalase compound II. UV/visible, MoĢssbauer, and X-ray absorption
spectroscopies have been used to examine the intermediate from pH
5 to 14. We have determined that HPC-II exists in an ironĀ(IV) hydroxide
state up to pH 11. Above this pH, the ironĀ(IV) hydroxide complex transitions
to a new species (p<i>K</i><sub>a</sub> = 13.1) with MoĢssbauer
parameters that are indicative of an ironĀ(IV)-oxo intermediate. Recently,
we discussed a role for an elevated compound II p<i>K</i><sub>a</sub> in diminishing the compound I reduction potential. This
has the effect of shifting the thermodynamic landscape toward the
two-electron chemistry that is critical for catalase function. In
catalase, a diminished potential would increase the selectivity for
peroxide disproportionation over off-pathway one-electron chemistry,
reducing the buildup of the inactive compound II state and reducing
the need for energetically expensive electron donor molecules
A 2.8 Ć FeāFe Separation in the Fe<sub>2</sub><sup>III/IV</sup> Intermediate, X, from <i>Escherichia coli</i> Ribonucleotide Reductase
A class
Ia ribonucleotide reductase (RNR) employs a Ī¼-oxo-Fe<sub>2</sub><sup>III/III</sup>/tyrosyl radical cofactor in its Ī²
subunit to oxidize a cysteine residue ā¼35 Ć
away in its
Ī± subunit; the resultant cysteine radical initiates substrate
reduction. During self-assembly of the <i>Escherichia coli</i> RNR-Ī² cofactor, reaction of the proteinās Fe<sub>2</sub><sup>II/II</sup> complex with O<sub>2</sub> results in accumulation
of an Fe<sub>2</sub><sup>III/IV</sup> cluster, termed <b>X</b>, which oxidizes the adjacent tyrosine (Y<sub>122</sub>) to the radical
(Y<sub>122</sub><sup>ā¢</sup>) as the cluster is converted to
the Ī¼-oxo-Fe<sub>2</sub><sup>III/III</sup> product. As the first
high-valent non-heme-iron enzyme complex to be identified and the
key activating intermediate of class Ia RNRs, <b>X</b> has been
the focus of intensive efforts to determine its structure. Initial
characterization by extended X-ray absorption fine structure (EXAFS)
spectroscopy yielded a FeāFe separation (<i>d</i><sub>FeāFe</sub>) of 2.5 Ć
, which was interpreted to
imply the presence of three single-atom bridges (O<sup>2ā</sup>, HO<sup>ā</sup>, and/or Ī¼-1,1-carboxylates). This short
distance has been irreconcilable with computational and synthetic
models, which all have <i>d</i><sub>FeāFe</sub> ā„
2.7 Ć
. To resolve this conundrum, we revisited the EXAFS characterization
of <b>X</b>. Assuming that samples containing increased concentrations
of the intermediate would yield EXAFS data of improved quality, we
applied our recently developed method of generating O<sub>2</sub> <i>in situ</i> from chlorite using the enzyme chlorite dismutase
to prepare <b>X</b> at ā¼2.0 mM, more than 2.5 times the
concentration realized in the previous EXAFS study. The measured <i>d</i><sub>FeāFe</sub> = 2.78 Ć
is fully consistent
with computational models containing a (Ī¼-oxo)<sub>2</sub>-Fe<sub>2</sub><sup>III/IV</sup> core. Correction of the <i>d</i><sub>FeāFe</sub> brings the experimental data and computational
models into full conformity and informs analysis of the mechanism
by which <b>X</b> generates Y<sub>122</sub><sup>ā¢</sup>
Characterization of a Cross-Linked ProteināNucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN
RlmN
and Cfr are methyltransferases/methylsynthases that belong
to the radical <i>S</i>-adenosylmethionine superfamily of
enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of
23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide,
and will subsequently catalyze C2 methylation if the site is unmethylated.
A key feature of the unusual mechanisms of catalysis proposed for
these enzymes is the attack of a methylene radical, derived from a
methylcysteine residue, onto the carbon center undergoing methylation
to generate a paramagnetic proteinānucleic acid cross-linked
species. This species has been thoroughly characterized during Cfr-dependent
C8 methylation, but does not accumulate to detectible levels in RlmN-dependent
C2 methylation. Herein, we show that inactive C118S/A variants of
RlmN accumulate a substrate-derived paramagnetic species. Characterization
of this species by electron paramagnetic resonance spectroscopy in
concert with strategic isotopic labeling shows that the radical is
delocalized throughout the adenine ring of A2503, although predominant
spin density is on N1 and N3. Moreover, <sup>13</sup>C hyperfine interactions
between the radical and the methylene carbon of the formerly [<i>methyl</i>-<sup>13</sup>C]ĀCys355 residue show that the radical
species exists in a covalent cross-link between the protein and the
nucleic acid substrate. X-ray structures of RlmN C118A show that,
in the presence of SAM, the substitution does not alter the active
site structure compared to that of the wild-type enzyme. Together,
these findings have new mechanistic implications for the role(s) of
C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement
of the residue in resolution of the cross-linked species via a radical
mediated process
Oxygen-Atom Transfer Reactivity of Axially Ligated Mn(V)āOxo Complexes: Evidence for Enhanced Electrophilic and Nucleophilic Pathways
Addition
of anionic donors to the manganeseĀ(V)āoxo corrolazine
complex Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz) has a dramatic influence
on oxygen-atom transfer (OAT) reactivity with thioether substrates.
The six-coordinate anionic [Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz)Ā(X)]<sup>ā</sup> complexes (X = F<sup>ā</sup>, N<sub>3</sub><sup>ā</sup>, OCN<sup>ā</sup>) exhibit a ā¼5
cm<sup>ā1</sup> downshift of the MnāO vibrational mode
relative to the parent Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz) complex
as seen by resonance Raman spectroscopy. Product analysis shows that
the oxidation of thioether substrates gives sulfoxide product, consistent
with single OAT. A wide range of OAT reactivity is seen for the different
axial ligands, with the following trend determined from a comparison
of their second-order rate constants for sulfoxidation: five-coordinate
ā thiocyanate ā nitrate < cyanate < azide <
fluoride āŖ cyanide. This trend correlates with DFT calculations
on the binding of the axial donors to the parent Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz) complex. A Hammett study was performed with <i>p</i>-X-C<sub>6</sub>H<sub>4</sub>SCH<sub>3</sub> derivatives and [Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz)Ā(X)]<sup>ā</sup> (X = CN<sup>ā</sup> or F<sup>ā</sup>) as the oxidant, and unusual
āV-shapedā Hammett plots were obtained. These results
are rationalized based upon a change in mechanism that hinges on the
ability of the [Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz)Ā(X)]<sup>ā</sup> complexes to function as either an electrophilic or weak nucleophilic
oxidant depending upon the nature of the <i>para</i>-X substituents.
For comparison, the one-electron-oxidized cationic Mn<sup>V</sup>(O)Ā(TBP<sub>8</sub>Cz<sup>ā¢+</sup>) complex yielded a linear Hammett relationship
for all substrates (Ļ = ā1.40), consistent with a straightforward
electrophilic mechanism. This study provides new, fundamental insights
regarding the influence of axial donors on high-valent Mn<sup>V</sup>(O) porphyrinoid complexes
O<sub>2</sub>-Evolving Chlorite Dismutase as a Tool for Studying O<sub>2</sub>-Utilizing Enzymes
The direct interrogation of fleeting intermediates by
rapid-mixing
kinetic methods has significantly advanced our understanding of enzymes
that utilize dioxygen. The gasās modest aqueous solubility
(<2 mM at 1 atm) presents a technical challenge to this approach,
because it limits the rate of formation and extent of accumulation
of intermediates. This challenge can be overcome by use of the heme
enzyme chlorite dismutase (Cld) for the rapid, <i>in situ</i> generation of O<sub>2</sub> at concentrations far exceeding 2 mM.
This method was used to define the O<sub>2</sub> concentration dependence
of the reaction of the class Ic ribonucleotide reductase (RNR) from <i>Chlamydia trachomatis</i>, in which the enzymeās Mn<sup>IV</sup>/Fe<sup>III</sup> cofactor forms from a Mn<sup>II</sup>/Fe<sup>II</sup> complex and O<sub>2</sub> via a Mn<sup>IV</sup>/Fe<sup>IV</sup> intermediate, at effective O<sub>2</sub> concentrations as high
as ā¼10 mM. With a more soluble receptor, myoglobin, an O<sub>2</sub> adduct accumulated to a concentration of >6 mM in <15
ms. Finally, the CāH-bond-cleaving Fe<sup>IV</sup>āoxo
complex, <b>J</b>, in taurine:Ī±-ketoglutarate dioxygenase
and superoxoāFe<sub>2</sub><sup>III/III</sup> complex, <b>G</b>, in <i>myo</i>-inositol oxygenase, and the tyrosyl-radical-generating
Fe<sub>2</sub><sup>III/IV</sup> intermediate, <b>X</b>, in <i>Escherichia coli</i> RNR, were all accumulated to yields more
than twice those previously attained. This means of <i>in situ</i> O<sub>2</sub> evolution permits a >5 mM āpulseā
of
O<sub>2</sub> to be generated in <1 ms at the easily accessible
Cld concentration of 50 Ī¼M. It should therefore significantly
extend the range of kinetic and spectroscopic experiments that can
routinely be undertaken in the study of these enzymes and could also
facilitate resolution of mechanistic pathways in cases of either sluggish
or thermodynamically unfavorable O<sub>2</sub> addition steps