The document summarizes research on the H134C mutant of the Thermus thermophilus Rieske protein, which substitutes one of the ligating histidines with a cysteine, altering the ligation structure of the iron-sulfur cluster. Spectroscopic analysis showed the mutant has a different pH dependence and stability compared to the wild type. Crystals of the mutant confirmed the 3-cysteine, 1-histidine ligation structure. Modification experiments with DEPC showed the mutant is modified but not reduced, and the modification may reverse over longer times. The results provide insights into proteins containing this type of iron-sulfur cluster ligation.

1. Spectroscopic Analysis and
Chemical Modification of the
H134C Mutant of the Thermus
thermophius Rieske Protein
Victor Rodríguez and Laura M. Hunsicker-Wang
Trinity University, San Antonio, TX

2. Abstract
The Rieske protein is found in the bc1 complex (complex III)
and plays an important role in transporting electrons and
protons through the electron transport chain. The Rieske
protein contains a [2Fe-2S] cluster ligated by a 2-cystines and
2- histidines. The reduction potential of this cluster is pH-
dependent and varies across species. An H134C mutant of
the Thermus thermophilus Rieskesubstitutes one of the
ligating histidines for a cysteine, changing the ligation
structure of the cluster from a 2Cys-2His to a 3Cys-1His
environment. To study the effects of this mutation, the protein
is subjected to modification with diethyl pyrocarbonate
(DEPC) and to pH changes. The behavior of this protein is
compared to the wild type protein as observed through
circular dichroism and UV visible spectroscopy. Because of the
similarity in the iron-sulfur cluster ligands, H134C will also be
compared to another mitochondrial protein, mitoNEET, which
contains a 3Cys-1His environment.

3. Rieske Proteins
• The Rieske protein is found in complex III
(cytochrome bc1 complex) as part of the
electron transport chain.
• This complex oxidizes ubiquinol to
ubiquinone and transports the electrons
through the complex into the Fe-S cluster in
Reiske.
http://www.scienceprofonline.org/metabolism/electron-transport-chain-cellular-respiration.html
http://en.wikipedia.org/wiki/Coenzyme_Q_%E2%80%93_cytochrome_c_reductase

4. H134C mutant & MitoNEET
• The Rieske protein Fe-S cluster has a 2-His 2-
Cys ligation environment.
• H134C mutant-the ligation structure is
altered to have a one histidine and three
cysteines.
• This ligation environment is similar to that of
MitoNEET, a known diabetes drug activator.
• Results from these experiments will be used
to compare to that of Mitoneet to better
understand the differences with this ligation
environment.
Rieske
MitoNEET
PDB ID 3REE

5. pH-Dependent UV-Visible spectra
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
300 400 500 600 700 800
Absorbance (A)
Wavelength (nm)
4.39
6.63
7.54
8.65
9.61
10.41
10.97
11.99
• Previous studies show that H134C has a large range of pH
stability
• H134C has a pKa of 9.86.
Data collected by Abhishek Chhetri
H134C

6. H134C Crystals
• Crystals for H134C Rieske
mutant were isolated under
the following conditions:
30% PEG 4000, 0.2 M
MgCl2, 0.1 M Tris-HCl pH
8.5 or 20% PEG 2000 MME,
0.01 M NiCl2· 6H2O, 0.1 M
Tris pH 8.5.
• Best data collected to 1.66
Å resolution!
• Space group P2221
Preliminary electron density map (2Fo-Fc)
showing 3 Cys, 1 His [2Fe2S] cluster
His 154
Cys 134
Cys 132 Cys 151

7. DEPC modification
• Deprotonated histidines react with DEPC.
• In truncated wild type, not only was modification observed, but
also reduction.
DEPC
DEPC = diethyl pyrocarbonate
Konkle et al. 2010 Biochemistry 49, 7272-7281

8. Reaction with DEPC- UV-Vis
pH 6
pH 7
pH 8
pH 6
pH 7
pH 8
Konkle et al. 2010 Biochemistry 49, 7272-7281
truncTtRp H120Q/H162Q
• truncTtRp and H120Q/H162Q are modified by DEPC
• Higher pH causes faster modification and more modification in truncTtRp

9. Effect of pH on DEPC reaction - CD
pH = 6 pH = 7
pH = 8 pH = 9
H120Q/H162Q
Konkle et al. 2010 Biochemistry 49, 7272-7281• More spectral changes with higher pH
• Proteins become reduced after modification

10. H134C Reaction with DEPC- UV
-0.05
0.05
0.15
0.25
0.35
0.45
0.55
200 300 400 500 600 700 800
Difference
Wavelength (nm)
pH 6.0
-0.05
0.05
0.15
0.25
0.35
0.45
0.55
200 300 400 500 600 700 800
Difference
Wavelength (nm)
pH 7.6
• H134C was reacted with DEPC
• difference UV-Vis spectra over 40 minutes at different pH values.
-0.05
0.05
0.15
0.25
0.35
0.45
0.55
200 300 400 500 600 700 800
Difference
Wavelelngth (nm)
pH 8.2
H134C is modified by DEPC, but the LMCT bands are minimally affected
-0.1
0
0.1
0.2
0.3
0.4
0.5
200 300 400 500 600 700 800
Difference
Wavelength (nm)
pH 9.0

11. pH-dependence of modification
• Reaction rate increases with pH, but the extent of modification decreases
above pH 7.6
• The profile differs from truncTtRp (see previous slide)
ΔAbs. 240 nm
Time (min)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 5 10 15 20 25
pH 6.0
pH 7.6
pH 8.2
pH 9.0

12. • H134C reacted with DEPC
over 90 minutes (upper
figure).
• A change in signal is
observed over time and
follow a similar patter to
what was observed for
reduction in wild type
Rieske.
• To fully understand the
changes of the signals, the
mutant protein was reduced
and oxidize (lower figure)
CD spectra change but the protein does not appear to become reduced
H134C Reaction with DEPC- CD

13. 24 hour reaction with DEPC
• The CD signal at 450 nm increases until 3 hours, and then
returns to the original level.
• One interpretation is that the DEPC modification reverses
over time, indicating a labile adduct.
-13
-11
-9
-7
-5
-3
-1
1
3
5
230 280 330 380 430 480 530 580
mdeg
Wavelength (nm)
H134C at pH 8.2 reacetd with DEPC for 24 hours
unreacted
0 hours
3 hours
10 hours
23 hours
27 hours
0
2
4
0 3 6 9 12 15 18 21 24 27
Absorbtion Time (hours)
H134C reacted with
DEPC at 450 nm

14. Conclusions
• H134C pKa of protein is 9.86.
• Crystal structure confirms the 3Cys 1 His
Structure
• Protein is modified by DEPC and not reduced
within a 90 minute reaction.
• DEPC modification may reverses over longer
times, indicating a labile adduct in this 3 Cys, 1
His ligation environment.

15. Acknowledgements
• McNair Scholar Program
• National Science Foundation
• P. John Hart Lab at the University of Texas
Health Science Center San Antonio for crystals