15 research outputs found
Functional Complementation Studies Reveal Different Interaction Partners of <i>Escherichia coli</i> IscS and Human NFS1
The
trafficking and delivery of sulfur to cofactors and nucleosides
is a highly regulated and conserved process among all organisms. All
sulfur transfer pathways generally have an l-cysteine desulfurase
as an initial sulfur-mobilizing enzyme in common, which serves as
a sulfur donor for the biosynthesis of sulfur-containing biomolecules
like iron–sulfur (Fe–S) clusters, thiamine, biotin,
lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides
in tRNA. The human l-cysteine desulfurase NFS1 and the <i>Escherichia coli</i> homologue IscS share a level of amino acid
sequence identity of ∼60%. While <i>E. coli</i> IscS
has a versatile role in the cell and was shown to have numerous interaction
partners, NFS1 is mainly localized in mitochondria with a crucial
role in the biosynthesis of Fe–S clusters. Additionally, NFS1
is also located in smaller amounts in the cytosol with a role in Moco
biosynthesis and mcm<sup>5</sup>s<sup>2</sup>U34 thio modifications
of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have
different interaction partners in their respective organisms. Here,
we used functional complementation studies of an <i>E. coli iscS</i> deletion strain with human NFS1 to dissect their conserved roles
in the transfer of sulfur to a specific target protein. Our results
show that human NFS1 and <i>E. coli</i> IscS share conserved
binding sites for proteins involved in Fe–S cluster assembly
like IscU, but not with proteins for tRNA thio modifications or Moco
biosynthesis. In addition, we show that human NFS1 was almost fully
able to complement the role of IscS in Moco biosynthesis when its
specific interaction partner protein MOCS3 from humans was also present
The Role of SufS Is Restricted to Fe–S Cluster Biosynthesis in <i>Escherichia coli</i>
In <i>Escherichia coli</i>, two different systems that
are important for the coordinate formation of Fe–S clusters
have been identified, namely, the ISC and SUF systems. The ISC system
is the housekeeping Fe–S machinery, which provides Fe–S
clusters for numerous cellular proteins. The IscS protein of this
system was additionally revealed to be the primary sulfur donor for
several sulfur-containing molecules with important biological functions,
among which are the molybdenum cofactor (Moco) and thiolated nucleosides
in tRNA. Here, we show that deletion of central components of the
ISC system in addition to IscS leads to an overall decrease in Fe–S
cluster enzyme and molybdoenzyme activity in addition to a decrease
in the number of Fe–S-dependent thiomodifications of tRNA,
based on the fact that some proteins involved in Moco biosynthesis
and tRNA thiolation are Fe–S-dependent. Complementation of
the ISC deficient strains with the <i>suf</i> operon restored
the activity of Fe–S-containing proteins, including the MoaA
protein, which is involved in the conversion of 5′GTP to cyclic
pyranopterin monophosphate in the fist step of Moco biosynthesis.
While both systems share a high degree of similarity, we show that
the function of their respective l-cysteine desulfurase IscS
or SufS is specific for each cellular pathway. It is revealed that
SufS cannot play the role of IscS in sulfur transfer for the formation
of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin,
being unable to interact with TusA or ThiI. The results demonstrate
that the role of the SUF system is exclusively restricted to Fe–S
cluster assembly in the cell
Low-Potential Amperometric Enzyme Biosensor for Xanthine and Hypoxanthine
The bacterial xanthine dehydrogenase
(XDH) from Rhodobacter capsulatus was
immobilized on an edge-plane
pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine
biosensor that functions at physiological pH. Phenazine methosulfate
(PMS) was used as a mediator which acts as an artificial electron-transfer
partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine
to xanthine and also xanthine to uric acid by an oxidative hydroxylation
mechanism. The present electrochemical biosensor was optimized in
terms of applied potential and pH. The electrocatalytic oxidation
response showed a linear dependence on the xanthine concentration
ranging from 1.0 × 10<sup>–5</sup> to 1.8 × 10<sup>–3</sup> M with a correlation coefficient of 0.994. The modified
electrode shows a very low detection limit for xanthine of 0.25 nM
(signal-to-noise ratio = 3) using controlled potential amperometry
Catalytic Electrochemistry of Xanthine Dehydrogenase
We report the mediated electrocatalytic voltammetry of
the molybdoenzyme
xanthine dehydrogenase (XDH) from <i>Rhodobacter capsulatus</i> at a thiol-modified Au electrode. The 2-electron acceptor <i>N</i>-methylphenazinium methanesulfonate (phenazine methosulfate,
PMS) is an effective artificial electron transfer partner for XDH
instead of its native electron acceptor NAD<sup>+</sup>. XDH catalyzes
the oxidative hydroxylation of hypoxanthine to xanthine and xanthine
to uric acid. Cyclic voltammetry was used to generate the active (oxidized)
form of the mediator. Simulation of the catalytic voltammetry across
a broad range of substrate and PMS concentrations at different sweep
rates was achieved with the program DigiSim to yield a set of consistent
rate and equilibrium constants that describe the catalytic system.
This provides the first example of the mediated electrochemistry of
a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference
from product oxidation. A remarkable two-step, sequential oxidation
of hypoxanthine to uric acid via xanthine by XDH is observed
Pyranopterin Dithiolene Distortions Relevant to Electron Transfer in Xanthine Oxidase/Dehydrogenase
The reducing substrates 4-thiolumazine
and 2,4-dithiolumazine have
been used to form Mo<sup>IV</sup>-product complexes with xanthine
oxidase (XO) and xanthine dehydrogenase. These Mo<sup>IV</sup>-product
complexes display an intense metal-to-ligand charge-transfer (MLCT)
band in the near-infrared region of the spectrum. Optical pumping
into this MLCT band yields resonance Raman spectra of the Mo site
that are devoid of contributions from the highly absorbing FAD and
2Fe2S clusters in the protein. The resonance Raman spectra reveal
in-plane bending modes of the bound product and low-frequency molybdenum
dithiolene and pyranopterin dithiolene vibrational modes. This work
provides keen insight into the role of the pyranopterin dithiolene
in electron-transfer reactivity
The N‑Terminus of Iron–Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with l‑Cysteine Desulfurase NFS1
Assembly
of iron–sulfur (FeS) clusters is an important process
in living cells. The initial sulfur mobilization step for FeS cluster
biosynthesis is catalyzed by l-cysteine desulfurase NFS1,
a reaction that is localized in mitochondria in humans. In humans,
the function of NFS1 depends on the ISD11 protein, which is required
to stabilize its structure. The NFS1/ISD11 complex further interacts
with scaffold protein ISCU and regulator protein frataxin, thereby
forming a quaternary complex for FeS cluster formation. It has been
suggested that the role of ISD11 is not restricted to its role in
stabilizing the structure of NFS1, because studies of single-amino
acid variants of ISD11 additionally demonstrated its importance for
the correct assembly of the quaternary complex. In this study, we
are focusing on the N-terminal region of ISD11 to determine the role
of N-terminal amino acids in the formation of the complex with NFS1
and to reveal the mitochondrial targeting sequence for subcellular
localization. Our in vitro studies with the purified proteins and
in vivo studies in a cellular system show that the first 10 N-terminal
amino acids of ISD11 are indispensable for the activity of NFS1 and
especially the conserved “LYR” motif is essential for
the role of ISD11 in forming a stable and active complex with NFS1
Functional Studies on <i>Oligotropha carboxidovorans</i> Molybdenum–Copper CO Dehydrogenase Produced in <i>Escherichia coli</i>
The
Mo/Cu-dependent CO dehydrogenase (CODH) from <i>Oligotropha
carboxidovorans</i> is an enzyme that is able to catalyze both
the oxidation of CO to CO<sub>2</sub> and the oxidation of H<sub>2</sub> to protons and electrons. Despite the close to atomic resolution
structure (1.1 Å), significant uncertainties have remained with
regard to the reaction mechanism of substrate oxidation at the unique
Mo/Cu center, as well as the nature of intermediates formed during
the catalytic cycle. So far, the investigation of the role of amino
acids at the active site was hampered by the lack of a suitable expression
system that allowed for detailed site-directed mutagenesis studies
at the active site. Here, we report on the establishment of a functional
heterologous expression system of <i>O. carboxidovorans</i> CODH in <i>Escherichia coli</i>. We characterize the purified
enzyme in detail by a combination of kinetic and spectroscopic studies
and show that it was purified in a form with characteristics comparable
to those of the native enzyme purified from <i>O. carboxidovorans</i>. With this expression system in hand, we were for the first time
able to generate active-site variants of this enzyme. Our work presents
the basis for more detailed studies of the reaction mechanism for
CO and H<sub>2</sub> oxidation of Mo/Cu-dependent CODHs in the future
Vibrational Probes of Molybdenum Cofactor–Protein Interactions in Xanthine Dehydrogenase
The pyranopterin
dithiolene (PDT) ligand is an integral component of the molybdenum
cofactor (Moco) found in all molybdoenzymes with the sole exception
of nitrogenase. However, the roles of the PDT in catalysis are still
unknown. The PDT is believed to be bound to the proteins by an extensive
hydrogen-bonding network, and it has been suggested that these interactions
may function to fine-tune Moco for electron- and atom-transfer reactivity
in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe
Moco–protein interactions using heavy-atom congeners of lumazine,
molecules that bind tightly to both wild-type xanthine dehydrogenase
(wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation
to the corresponding violapterin product molecules. The resulting
enzyme–product complexes possess intense near-IR absorption,
allowing high-quality rR spectra to be collected on wt-XDH and the
Q102G and Q197A variants. Small negative frequency shifts relative
to wt-XDH are observed for the low-frequency Moco vibrations. These
results are interpreted in the context of weak hydrogen-bonding and/or
electrostatic interactions between Q102 and the −NH<sub>2</sub> terminus of the PDT, and between Q197 and the terminal oxo of the
MoO group. The Q102A, Q102G, Q197A, and Q197E variants do
not appreciably affect the kinetic parameters <i>k</i><sub>red</sub> and <i>k</i><sub>red</sub>/<i>K</i><sub>D</sub>, indicating that a primary role for these glutamine
residues is to stabilize and coordinate Moco in the active site of
XO family enzymes but to not directly affect the catalytic throughput.
Raman frequency shifts between wt-XDH and its Q102G variant suggest
that the changes in the electron density at the Mo ion that accompany
Mo oxidation during electron-transfer regeneration of the catalytically
competent active site are manifest in distortions at the distant PDT
amino terminus. This implies a primary role for the PDT as a conduit
for facilitating enzymatic electron-transfer reactivity in xanthine
oxidase family enzymes
Complementary Surface-Enhanced Resonance Raman Spectroscopic Biodetection of Mixed Protein Solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles
Silver nanoparticles with identical plasmonic properties
but different
surface functionalities are synthesized and tested as chemically selective
surface-enhanced resonance Raman (SERR) amplifiers in a two-component
protein solution. The surface plasmon resonances of the particles
are tuned to 413 nm to match the molecular resonance of protein heme
cofactors. Biocompatible functionalization of the nanoparticles with
a thin film of chitosan yields selective SERR enhancement of the anionic
protein cytochrome <i>b</i><sub>5</sub>, whereas functionalization
with SiO<sub>2</sub> amplifies only the spectra of the cationic protein
cytochrome <i>c</i>. As a result, subsequent addition of
the two differently functionalized particles yields complementary
information on the same mixed protein sample solution. Finally, the
applicability of chitosan-coated Ag nanoparticles for protein separation
was tested by in situ resonance Raman spectroscopy
Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X‑ray Absorption Spectroscopy
Enzymes
of the xanthine oxidase family are among the best characterized mononuclear
molybdenum enzymes. Open questions about their mechanism of transfer
of an oxygen atom to the substrate remain. The enzymes share a molybdenum
cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule
via its dithiolene function and terminal sulfur and oxygen groups.
For xanthine dehydrogenase (XDH) from the bacterium <i>Rhodobacter
capsulatus</i>, we used X-ray absorption spectroscopy to determine
the Mo site structure, its changes in a pH range of 5–10, and
the influence of amino acids (Glu730 and Gln179) close to Moco in
wild-type (WT), Q179A, and E730A variants, complemented by enzyme
kinetics and quantum chemical studies. Oxidized WT and Q179A revealed
a similar Mo(VI) ion with each one MPT, MoO, Mo–O<sup>–</sup>, and MoS ligand, and a weak Mo–O(E730)
bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand
(p<i>K</i> ∼ 6.8) causes inhibition of XDH at acidic
pH, whereas deprotonated xanthine (p<i>K</i> ∼ 8.8)
is an inhibitor at alkaline pH. A similar acidic p<i>K</i> for the WT and Q179A variants, as well as the metrical parameters
of the Mo site and density functional theory calculations, suggested
protonation at the equatorial oxo group. The sulfido was replaced
with an oxo ligand in the inactive E730A variant, further showing
another oxo and one Mo–OH ligand at Mo, which are independent
of pH. Our findings suggest a reaction mechanism for XDH in which
an initial oxo rather than a hydroxo group and the sulfido ligand
are essential for xanthine oxidation