7 research outputs found
A Single Outer-Sphere Mutation Stabilizes apo-Mn Superoxide Dismutase by 35 °C and Disfavors Mn Binding
The catalytic active site of Mn-specific
superoxide dismutase (MnSOD)
is organized around a redox-active Mn ion. The most highly conserved
difference between MnSODs and the homologous FeSODs is the origin
of a Gln in the second coordination sphere. In MnSODs it derives from
the C-terminal domain whereas in FeSODs it derives from the N-terminal
domain, yet its side chain occupies almost superimposable positions
in the active sites of these two types of SODs. Mutation of this Gln69
to Glu in Escherichia coli FeSOD increased
the Fe<sup>3+/2+</sup> reduction midpoint potential by >0.6 V without
disrupting the structure or Fe binding [Yikilmaz, E., Rodgers, D. W., and Miller,
A.-F. (2006) Biochemistry 45 (4), 1151−1161]. We now describe the analogous Q146E mutant of MnSOD,
explaining its low Mn content in terms increased stability of the
apo-Mn protein. In 0.8 M guanidinium HCl, Q146E-apoMnSOD displays
an apparent melting midpoint temperature (<i>T</i><sub>m</sub>) 35 °C <i>higher</i> that of wild-type (WT) apoMnSOD,
whereas the <i>T</i><sub>m</sub> of WT-holoMnSOD is only
20 °C higher than that of WT-apoMnSOD. In contrast, the <i>T</i><sub>m</sub> attributed to Q146E-holoMnSOD is 40 °C <i>lower</i> than that of Q146E-apoMnSOD. Thus, our data refute
the notion that the WT residues optimize the structural stability
of the protein and instead are consistent with conservation on the
basis of enzyme function and therefore ability to bind metal ion.
We propose that the WT-MnSOD protein conserves a destabilizing amino
acid at position 146 as part of a strategy to favor metal ion binding
Two Major Pre-Nucleation Species that are Conformationally Distinct and in Equilibrium of Self-Association
To
understand how solution chemistry governs polymorphic formation
of organic crystals, solution NMR measurements of tolfenamic acid
were conducted in ethanol. It was unveiled by chemical shift and diffusivity
results that the solute molecules self-associated as dimers in solution.
Further nOe (nuclear Overhauser effect) analyses indicate that a more
twisted conformation became dominant over a planar conformation under
the solution conditions that favored the dimer formation. This discovery
is rationalized in terms of the energy balance between the conformation
and intermolecular hydrogen bonding of the solute molecule, suggesting
a significant role of the cooperability between a molecule’s
conformation and its intermolecular interaction in determining the
nucleation outcome of distinct crystal structures
<sup>1</sup>H Dynamic Nuclear Polarization Based on an Endogenous Radical
We demonstrate a 15-fold enhancement of solid-state NMR signals
via dynamic nuclear polarization (DNP) based on a stable, naturally
occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone
of flavodoxin. The line width of flavodoxin’s EPR signal suggests
that the dominant DNP mechanism is the solid effect, consistent with
the field-dependent DNP enhancement profile. The magnitude of the
enhancement as well as the bulk-polarization build-up time constant
(Ï„<sub>B</sub>) with which it develops are dependent on the
isotopic composition of the protein. Deuteration of the protein to
85% increased the nuclear longitudinal relaxation time <i>T</i><sub>1n</sub> and Ï„<sub>B</sub> by factors of five and seven,
respectively. Slowed dissipation of polarization can explain the 2-fold
higher maximal enhancement than that obtained in proteated protein,
based on the endogenous semiquinone. In contrast, the long Ï„<sub>B</sub> of TOTAPOL-based DNP in nonglassy samples was not accompanied
by a similarly important long <i>T</i><sub>1n</sub>, and
in this case the enhancement was greatly reduced. The low concentrations
of radicals occurring naturally in biological systems limit the magnitude
of DNP enhancement that is attainable by this means. However, our
enhancement factors of up to 15 can nonetheless make an important
difference to the feasibility of applying solid-state NMR to biochemical
systems. We speculate that DNP based on endogenous radicals may facilitate
MAS NMR characterization of biochemical complexes and even organelles,
and could also serve as a source of additional structural and physiological
information
Geometric and Electronic Structures of Manganese-Substituted Iron Superoxide Dismutase
The
active-site structures of the oxidized and reduced forms of manganese-substituted
iron superoxide dismutase (MnÂ(Fe)ÂSOD) are examined, for the first
time, using a combination of spectroscopic and computational methods.
On the basis of electronic absorption, circular dichroism (CD), magnetic
CD (MCD), and variable-temperature variable-field MCD data obtained
for oxidized MnÂ(Fe)ÂSOD, we propose that the active site of this species
is virtually identical to that of wild-type manganese SOD (MnSOD),
with both containing a metal ion that resides in a trigonal bipyramidal
ligand environment. This proposal is corroborated by quantum mechanical/molecular
mechanical (QM/MM) computations performed on complete protein models
of MnÂ(Fe)ÂSOD in both its oxidized and reduced states and, for comparison,
wild-type (WT) MnSOD. The major differences between the QM/MM optimized
active sites of WT MnSOD and MnÂ(Fe)ÂSOD are a smaller (His)ÂN–Mn–NÂ(His)
equatorial angle and a longer (Gln146(69))ÂNH···OÂ(sol)
H-bond distance in the metal-substituted protein. Importantly, these
modest geometric differences are consistent with our spectroscopic
data obtained for the oxidized proteins and high-field electron paramagnetic
resonance spectra reported previously for reduced MnÂ(Fe)ÂSOD and MnSOD.
As MnÂ(Fe)ÂSOD exhibits a reduction midpoint potential (<i>E</i><sub>m</sub>) almost 700 mV higher than that of MnSOD, which has
been shown to be sufficient for explaining the lack of SOD activity
displayed by the metal-subtituted species (Vance, C. K.; Miller, A.
F. <i>Biochemistry</i> <b>2001</b>, <i>40</i>, 13079–13087), <i>E</i><sub>m</sub>’s were
computed for our experimentally validated QM/MM optimized models of
MnÂ(Fe)ÂSOD and MnSOD. These computations properly reproduce the experimental
trend and reveal that the drastically elevated <i>E</i><sub>m</sub> of the metal substituted protein stems from a larger separation
between the second-sphere Gln residue and the coordinated solvent
in MnÂ(Fe)ÂSOD relative to MnSOD, which causes a weakening of the corresponding
H-bond interaction in the oxidized state and alleviates steric crowding
in the reduced state
Multitechnique Investigation of the pH Dependence of Phosphate Induced Transformations of ZnO Nanoparticles
In order to properly evaluate the
ecological and human health risks
of ZnO manufactured nanomaterials (MNMs) released to the environment,
it is critical to understand the likely transformation products in
various environments, such as soils, surface and ground waters, and
wastewater treatment processes. To address this knowledge gap, we
examined the transformation of 30 nm ZnO MNMs in the presence of different
concentrations of phosphate as a function of time and pH using a variety
of orthogonal analytical techniques. The data reveal that ZnO MNMs
react with phosphate at various concentrations and transform into
two distinct morphological/structural phases: a micrometer scale crystalline
zinc phosphate phase (hopeite-like) and a nanoscale phase that likely
consists of a ZnO core with an amorphous Zn<sub>3</sub>(PO<sub>4</sub>)<sub>2</sub> shell. The P species composition was also pH dependent,
with 82% occurring as hopeite-like P at pH 6 while only 15% occurred
as hopeite-like P at pH 8. These results highlight how reactions of
ZnO MNMs with phosphate are influenced by environmental variables,
including pH, and may ultimately result in structurally and morphologically
heterogeneous end products
The Electron Bifurcating FixABCX Protein Complex from <i>Azotobacter vinelandii</i>: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis
The biological reduction of dinitrogen
(N<sub>2</sub>) to ammonia
(NH<sub>3</sub>) by nitrogenase is an energetically demanding reaction
that requires low-potential electrons and ATP; however, pathways used
to deliver the electrons from central metabolism to the reductants
of nitrogenase, ferredoxin or flavodoxin, remain unknown for many
diazotrophic microbes. The FixABCX protein complex has been proposed
to reduce flavodoxin or ferredoxin using NADH as the electron donor
in a process known as electron bifurcation. Herein, the FixABCX complex
from <i>Azotobacter vinelandii</i> was purified and demonstrated
to catalyze an electron bifurcation reaction: oxidation of NADH (<i>E</i><sub>m</sub> = −320 mV) coupled to reduction of
flavodoxin semiquinone (<i>E</i><sub>m</sub> = −460
mV) and reduction of coenzyme Q (<i>E</i><sub>m</sub> =
10 mV). Knocking out <i>fix</i> genes rendered Δ<i>rnf A. vinelandii</i> cells unable to fix dinitrogen, confirming
that the FixABCX system provides another route for delivery of electrons
to nitrogenase. Characterization of the purified FixABCX complex revealed
the presence of flavin and iron–sulfur cofactors confirmed
by native mass spectrometry, electron paramagnetic
resonance spectroscopy, and transient absorption spectroscopy. Transient
absorption spectroscopy further established the presence of a short-lived
flavin semiquinone radical, suggesting that a thermodynamically unstable
flavin semiquinone may participate as an intermediate in the transfer
of an electron to flavodoxin. A structural model of FixABCX, generated
using chemical cross-linking in conjunction with homology modeling,
revealed plausible electron transfer pathways to both high- and low-potential
acceptors. Overall, this study informs a mechanism for electron bifurcation,
offering insight into a unique method for delivery of low-potential
electrons required for energy-intensive biochemical conversions