The protein samples used in electron
transfer and in vitro cysteine desulfurase assay
and Fe–S cluster reconstitution experiments were prepared in
an anaerobic
chamber (Coy Laboratory) with samples buffer-exchanged extensively
with anaerobic HN buffer prior to the experiments. The reaction volumes
in all the experiments were kept to 1 mL. A UV-1700 UV/visible spectrophotometer
(Shimadzu, Kyoto, Japan)
with a temperature control unit was used to collect the spectra, and
UVProbe version 2.21 (Shimadzu) was used to collect and analyze the
data.
Electron transfer from re-FDX1 or re-FDX2 to the [Acp]2:[ISD11]2:[NFS1]2 complex was monitored
as follows. re-FDX1 or re-FDX2 (25 μM) was mixed with 25 μM
[Acp]2:[ISD11]2:[NFS1]2, and 125
μMl -cysteine was added to initiate the reaction. Samples
were then transferred
to 1 cm path-length quartz cuvettes, sealed with rubber septa, and
UV/vis
spectra were collected at 25 °C. Control experiments withoutl -cysteine were also conducted.
The cysteine desulfurase
assay reaction mixtures (300 μL in HN buffer) contained 1 μM
[Acp]2:[ISD11]2:[NFS1]2 and 50 μMl -cysteine. The reductant was 10 μM re-FDX1, 10 μM
re-FDX2, 10 μM DTT, or 1 mM DTT. Thel -cysteine was
added
last to initiate the reaction. One or more of
the following components were added to assess their effects on sulfide
production: 10 μM ISCU, 10 μM FXN, and 50 μM Fe2(NH4)2(SO4)2.
After anaerobic incubation for 20 min at room temperature, the
reaction mixture was diluted to 800 μL, and 100 μL of
20 mM N,N-dimethyl-p-phenylenediamine in 7.2 M HCl and 100 μL of 30 mM FeCl3 in 1.2 M HCl were added to quench the reaction and convert
sulfide to methylene blue. The quenched reaction mixture was incubated
for 15 min at room temperature, and then the absorbance at 670 nm
was measured and used to estimate the amount of sulfide by comparison
to a standard curve obtained from known concentrations of Na2S.
The in vitro Fe–S cluster reconstitution
assays were performed as follows. Reaction
mixtures (1 mL) prepared in the anaerobic chamber contained 25 μM
re-FDX1 or re-FDX2, 0.5 μM [Acp]2:[ISD11]2:[NFS1]2, 25 μM ISCU, and 125 μM Fe2(NH4)2(SO4)2.l -cysteine (final concentration of 125 μM) was added to initiate
the experiment. Samples were then transferred
to 1 cm path-length quartz cuvettes, sealed with rubber septa, and
UV/vis
spectra were collected at 25 °C. Control experiments were conducted
withoutl -cysteine.
transfer and in vitro cysteine desulfurase assay
and Fe–S cluster reconstitution experiments were prepared in
an anaerobic
chamber (Coy Laboratory) with samples buffer-exchanged extensively
with anaerobic HN buffer prior to the experiments. The reaction volumes
in all the experiments were kept to 1 mL. A UV-1700 UV/visible spectrophotometer
(Shimadzu, Kyoto, Japan)
with a temperature control unit was used to collect the spectra, and
UVProbe version 2.21 (Shimadzu) was used to collect and analyze the
data.
Electron transfer from re-FDX1 or re-FDX2 to the [Acp]2:[ISD11]2:[NFS1]2 complex was monitored
as follows. re-FDX1 or re-FDX2 (25 μM) was mixed with 25 μM
[Acp]2:[ISD11]2:[NFS1]2, and 125
μM
were then transferred
to 1 cm path-length quartz cuvettes, sealed with rubber septa, and
UV/vis
spectra were collected at 25 °C. Control experiments without
The cysteine desulfurase
assay reaction mixtures (300 μL in HN buffer) contained 1 μM
[Acp]2:[ISD11]2:[NFS1]2 and 50 μM
re-FDX2, 10 μM DTT, or 1 mM DTT. The
added
last to initiate the reaction. One or more of
the following components were added to assess their effects on sulfide
production: 10 μM ISCU, 10 μM FXN, and 50 μM Fe2(NH4)2(SO4)2.
After anaerobic incubation for 20 min at room temperature, the
reaction mixture was diluted to 800 μL, and 100 μL of
20 mM N,N-dimethyl-p-phenylenediamine in 7.2 M HCl and 100 μL of 30 mM FeCl3 in 1.2 M HCl were added to quench the reaction and convert
sulfide to methylene blue. The quenched reaction mixture was incubated
for 15 min at room temperature, and then the absorbance at 670 nm
was measured and used to estimate the amount of sulfide by comparison
to a standard curve obtained from known concentrations of Na2S.
The in vitro Fe–S cluster reconstitution
assays were performed as follows. Reaction
mixtures (1 mL) prepared in the anaerobic chamber contained 25 μM
re-FDX1 or re-FDX2, 0.5 μM [Acp]2:[ISD11]2:[NFS1]2, 25 μM ISCU, and 125 μM Fe2(NH4)2(SO4)2.
the experiment. Samples were then transferred
to 1 cm path-length quartz cuvettes, sealed with rubber septa, and
UV/vis
spectra were collected at 25 °C. Control experiments were conducted
without
