5 research outputs found
Cytosolic Fe-S cluster protein maturation and iron regulation are independent of the mitochondrial Erv1/Mia40 import system
The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways
Structural Context Effects in the Oxidation of 8‑Oxo-7,8-dihydro-2′-deoxyguanosine to Hydantoin Products: Electrostatics, Base Stacking, and Base Pairing
8-Oxo-7,8-dihydroguanine (OG) is the most common base
damage found
in cells, where it resides in many structural contexts, including
the nucleotide pool, single-stranded DNA at transcription forks and
replication bubbles, and duplex DNA base-paired with either adenine
(A) or cytosine (C). OG is prone to further oxidation to the highly
mutagenic hydantoin products spiroiminodihydantoin (Sp) and 5-guanidinohydantoin
(Gh) in a sharply pH-dependent fashion within nucleosides. In the
present work, studies were conducted to determine how the structural
context affects OG oxidation to the hydantoins. These studies revealed
a trend in which the Sp yield was greatest in unencumbered contexts,
such as nucleosides, while the Gh yield increased in oligodeoxynucleotide
(ODN) contexts or at reduced pH. Oxidation of oligomers containing
hydrogen-bond modulators (2,6-diaminopurine, <i>N</i><sup><i>4</i></sup>-ethylcytidine) or alteration of the reaction
conditions (pH, temperature, and salt) identify base stacking, electrostatics,
and base pairing as the drivers of the key intermediate 5-hydroxy-8-oxo-7,8-dihydroguanine
(5-HO-OG) partitioning along the two hydantoin pathways, allowing
us to propose a mechanism for the observed base-pairing effects. Moreover,
these structural effects cause an increase in the effective p<i>K</i><sub>a</sub> of 5-HO-OG, following an increasing trend
from 5.7 in nucleosides to 7.7 in a duplex bearing an OG·C base
pair, which supports the context-dependent product yields. The high
yield of Gh in ODNs underscores the importance of further study on
this lesion. The structural context of OG also determined its relative
reactivity toward oxidation, for which the OG·A base pair is
∼2.5-fold more reactive than an OG·C base pair, and with
the weak one-electron oxidant ferricyanide, the OG nucleoside reactivity
is >6000-fold greater than that of OG·C in a duplex, leading
to the conclusion that OG in the nucleoside pool should act as a protective
agent for OG in the genome
The <i>Escherichia coli</i> BolA Protein IbaG Forms a Histidine-Ligated [2Fe-2S]-Bridged Complex with Grx4
Two
ubiquitous protein families have emerged as key players in
iron metabolism, the CGFS-type monothiol glutaredoxins (Grxs) and
the BolA proteins. Monothiol Grxs and BolA proteins form heterocomplexes
that have been implicated in Fe–S cluster assembly and trafficking.
The <i>Escherichia coli</i> genome encodes members of both
of these proteins families, namely, the monothiol glutaredoxin Grx4
and two BolA family proteins, BolA and IbaG. Previous work has demonstrated
that <i>E. coli</i> Grx4 and BolA interact as both apo and
[2Fe-2S]-bridged heterodimers that are spectroscopically distinct
from [2Fe-2S]-bridged Grx4 homodimers. However, the physical and functional
interactions between Grx4 and IbaG are uncharacterized. Here we show
that co-expression of Grx4 with IbaG yields a [2Fe-2S]-bridged Grx4–IbaG
heterodimer. <i>In vitro</i> interaction studies indicate
that IbaG binds the [2Fe-2S] Grx4 homodimer to form apo Grx4–IbaG
heterodimer as well as the [2Fe-2S] Grx4–IbaG heterodimer,
altering the cluster stability and coordination environment. Additionally,
spectroscopic and mutagenesis studies provide evidence that IbaG ligates
the Fe–S cluster via the conserved histidine that is present
in all BolA proteins and by a second conserved histidine that is present
in the H/C loop of two of the four classes of BolA proteins. These
results suggest that IbaG may function in Fe–S cluster assembly
and trafficking in <i>E. coli</i> as demonstrated for other
BolA homologues that interact with monothiol Grxs