4 research outputs found
Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human FeāS Assembly Complex
Ironāsulfur
(FeāS) clusters function as protein cofactors
for a wide variety of critical cellular reactions. In human mitochondria,
a core FeāS assembly complex [called SDUF and composed of NFS1,
ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes FeāS
clusters from iron, cysteine sulfur, and reducing equivalents and
then transfers these intact clusters to target proteins. <i>In
vitro</i> assays have relied on reducing the complexity of this
complicated FeāS assembly process by using surrogate electron
donor molecules and monitoring simplified reactions. Recent studies
have concluded that FXN promotes the synthesis of [4Fe-4S] clusters
on the mammalian FeāS assembly complex. Here the kinetics of
FeāS synthesis reactions were determined using different electron
donation systems and by monitoring the products with circular dichroism
and absorbance spectroscopies. We discovered that common surrogate
electron donor molecules intercepted FeāS cluster intermediates
and formed high-molecular weight species (HMWS). The HMWS are associated
with iron, sulfide, and thiol-containing proteins and have properties
of a heterogeneous solubilized mineral with spectroscopic properties
remarkably reminiscent of those of [4Fe-4S] clusters. In contrast,
reactions using physiological reagents revealed that FXN accelerates
the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as
previously reported. In the preceding paper [Fox, N. G., et al. (2015) <i>Biochemistry 54</i>, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates
on the SDUF complex were shown to readily transfer to uncomplexed
ISCU2 or apo acceptor proteins, depending on the reaction conditions.
Our results indicate that FXN accelerates a rate-limiting sulfur transfer
step in the synthesis of [2Fe-2S] clusters on the human FeāS
assembly complex
Human Frataxin Activates FeāS Cluster Biosynthesis by Facilitating Sulfur Transfer Chemistry
Ironāsulfur clusters are ubiquitous
protein cofactors with
critical cellular functions. The mitochondrial FeāS assembly
complex, which consists of the cysteine desulfurase NFS1 and its accessory
protein (ISD11), the FeāS assembly protein (ISCU2), and frataxin
(FXN), converts substrates l-cysteine, ferrous iron, and
electrons into FeāS clusters. The physiological function of
FXN has received a tremendous amount of attention since the discovery
that its loss is directly linked to the neurodegenerative disease
Friedreichās ataxia. Previous <i>in vitro</i> results
revealed a role for human FXN in activating the cysteine desulfurase
and FeāS cluster biosynthesis activities of the FeāS
assembly complex. Here we present radiolabeling experiments that indicate
FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting
persulfide species is viable in the subsequent synthesis of FeāS
clusters. Additional mutagenesis, enzyme kinetic, UVāvisible,
and circular dichroism spectroscopic studies suggest conserved ISCU2
residue C104 is critical for FXN activation, whereas C35, C61, and
C104 are all essential for FeāS cluster formation on the assembly
complex. These results cannot be fully explained by the hypothesis
that FXN functions as an iron donor for FeāS cluster biosynthesis,
and further support an allosteric regulator role for FXN. Together,
these results lead to an activation model in which FXN accelerates
persulfide formation on NFS1 and favors a helix-to-coil interconversion
on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2
as an initial step in FeāS cluster biosynthesis
Fluorescent Probes for Tracking the Transfer of IronāSulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways
Ironāsulfur
(FeāS) clusters are protein cofactors
that are constructed and delivered to target proteins by elaborate
biosynthetic machinery. Mechanistic insights into these processes
have been limited by the lack of sensitive probes for tracking FeāS
cluster synthesis and transfer reactions. Here we present fusion protein-
and intein-based fluorescent labeling strategies that can probe FeāS
cluster binding. The fluorescence is sensitive to different cluster
types ([2Feā2S] and [4Feā4S] clusters), ligand environments
([2Feā2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin),
and cluster oxidation states. The power of this approach is highlighted
with an extreme example in which the kinetics of FeāS cluster
transfer reactions are monitored between two Fdx molecules that have
identical FeāS spectroscopic properties. This exchange reaction
between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT),
a result that was confirmed by mass spectrometry. DTT likely functions
in a ligand substitution reaction that generates a [2Feā2S]āDTT
species, which can transfer the cluster to either labeled or unlabeled
Fdx. The ability to monitor this challenging cluster exchange reaction
indicates that real-time FeāS cluster incorporation can be
tracked for a specific labeled protein in multicomponent assays that
include several unlabeled FeāS binding proteins or other chromophores.
Such advanced kinetic experiments are required to untangle the intricate
networks of transfer pathways and the factors affecting flux through
branch points. High sensitivity and suitability with high-throughput
methodology are additional benefits of this approach. We anticipate
that this cluster detection methodology will transform the study of
FeāS cluster pathways and potentially other metal cofactor
biosynthetic pathways
Molecular Engineering of Organophosphate Hydrolysis Activity from a Weak Promiscuous Lactonase Template
Rapid evolution of enzymes provides
unique molecular insights into the remarkable adaptability of proteins
and helps to elucidate the relationship between amino acid sequence,
structure, and function. We interrogated the evolution of the phosphoĀtriesterase
from Pseudomonas diminuta (<i>Pd</i>PTE), which hydrolyzes synthetic organophosphates with
remarkable catalytic efficiency. PTE is thought to be an evolutionarily
āyoungā enzyme, and it has been postulated that it has
evolved from members of the phosphoĀtriesterase-like lactonase
(PLL) family that show promiscuous organophosphate-degrading activity.
Starting from a weakly promiscuous PLL scaffold (<i>Dr</i>0930 from Deinococcus radiodurans),
we designed an extremely efficient organophosphate hydrolase (OPH)
with broad substrate specificity using rational and random mutagenesis
in combination with in vitro activity screening. The OPH activity
for seven organophosphate substrates was simultaneously enhanced by
up to 5 orders of magnitude, achieving absolute values of catalytic
efficiencies up to 10<sup>6</sup> M<sup>ā1</sup> s<sup>ā1</sup>. Structural and computational analyses identified the molecular
basis for the enhanced OPH activity of the engineered PLL variants
and demonstrated that OPH catalysis in <i>Pd</i>PTE and
the engineered PLL differ significantly in the mode of substrate binding