Characterization of the H134C mutant of the Thermus thermophilus Rieske ProteinVictor Rodríguez, Nathan Webber and Laura Hunsicker-WangDepartment of Chemistry, Trinity University, San Antonio, TX

PRELIMINARY H134C CRYSTAL STRUCTUREABSTRACT

The Rieske protein is found in the bc1 complex of the electron transport chain, playing an important role in transporting electrons and protons . It contains a [2Fe-2S] clus ter ligated by two cys teines and two his tidines . The reduction potential of this clus ter is pH-dependent and varies across species . The H134C mutant of the Thermus thermophilus Rieske protein subs titutes one of the ligating his tidines for a cys teine, changing the ligation s tructure of the clus ter from a 2Cys-2His ligation environment to a 3Cys-1His environment. To s tudy the effects of this mutation, the protein was subjected to modification with diethyl pyrocarbonate (DEPC) at varying pH values . The behavior of this protein was observed and compared to a truncated form of the wild type protein through circular dichroism and UV vis ible spectroscopy. DEPC modification was observed in both wild type and H134C protein. H134C was also subjected to β-Mercaptoethanol and thiazolidinedione, which showed minimal change over time. In contras t, wild type Rieske showed lots of spectral change when exposed to β-Mercaptoethanol. Because of the s imilarity in the iron-sulfur clus ter ligands , H134C was also compared to another mitochondrial protein, mitoNEET, which contains a 3Cys-1His environment and has been class ified as a diabetes drug activator. MitoNEET has been previous ly shown to be uns table at lower pH values , in contras t to H134C, which is s table at both low and high pH. By comparing what is s imilar and what is different in the pH and chemical reactivity of H134C and MitoNEET, we can decipher what are common characteris tics of the 3Cys-1His environment and what are unique properties of the different proteins .

Figure 1. (a) Rieske proteins are found in complex III (cytochrome bc1

complex) of the electron transport chain. (b) Rieske plays an important role in the Q cycle, which transports electrons through ETC and pump H+

into mitochondrial matrix .

RIESKE PROTEINS

(a) (b)

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h ttp ://en.wikip edia.org/wiki/File:Complex_ III.png

RIESKE STRUCTURE

Figure 2. Crystal structure of truncated wild type Rieske protein from Thermus thermophilus. Rieske has a [2Fe-2S] cluster that is ligated by 2 histidines and 2 cysteines. PDB: 3FOU

• Rieske proteins have a large range of reduction potentials that vary across species.

pH-DEPENDENT REDUCTION POTENTIAL

Zu et al. J Am. Chem. Soc., 2001 123, 9906-9907

Figure 3. pH-dependent reduction potential plot for the Rieske protein from Thermus thermophilus. • Rieske proteins have two 2 pKas in

its oxidized and reduced state due to the two ligating histidines (pKox1

7.85 ± 0.1, pKo x 2 9 .65 ± 0.1,

pKred 1 ,2 12.5 ± 0.2). • These reduction potentials can be

altered by the number of hydrogen bonds, the proximity of charged residues, and solvent accessibility to the cluster.

H134C

His 134

His 154

Cys 134

His 154

Figure 4. Rieske cluster changed from a 2 Cys – 2 His ligation environment to a 3 Cys – 1 His. The H134C mutant of Rieske protein from Thermus thermophilus was created to probe how a change in the ligation environment around the cluster affects its reduction potential.

His tidine

Cys teine

Cys teine

Cys teine

Figure 6. Preliminary crystal structure of the H134C Rieske mutant. 2Fo – Fc

electron density map contoured at 1 .5 sigma shows the 3Cys-1His ligation environment around the [2Fe-2S] cluster. Current statistical crystal data: R: 0 .3478, R-free: 0 .3502,

MitoNEET

Figure 5. Structure of MitoNEET. The figure shows a monomer of the normally dimeric protein. MitoNEET is a mitochondrial membrane protein that has been identified as a diabetes activating drug. It has a similar ligation environment to the H134C Rieske mutant. Results from these H134C experiments will be used to compare to MitoNEET to better understand the differences with this ligation environment. PDB 3REE

pH DEPENDENT UV-VISIBLE SPECTRA*

Figure 8. (a) UV-Visible spectra of H134C at different pH values. (b) Titration curve for H134C at 333 nm which was fit and a pKo xof 9 .86 was obtained.* Data collected by Abhishek Chhetri

(a)

(b)

H134C STABILITY

Figure 7. H134C stability over different pH values over time. There are nonoticeable changes in the spectra of H134C after letting it sit for 40 minutes (normal reaction times) at different pH values, showing the stability of H134C, which differs from MitoNEET.

pH-DEPENDENT CIRCULAR DICROISM SPECTRA

Figure 9. (a) Circular dichroism (CD) spectra of H134C at different pH values. (b) Titration curve for H134C at 324 nm., 392 nm, 445 nm, and 481 nm. Changes in spectra at these wavelengths reveal a pKo x

for H134C ranging from 9.33 – 9.87, which is similar to UV-Visible data.

(a)(b)

DEPC

DEPC MODIFICATION

Figure 10. Reaction of Rieske protein histidines with diethyl pyrocarbonate (DEPC). Deprotonated histidines are able to react with DEPC. In truncated wild type Rieske, not only was modification observed, but also reduction.

H134C REACTION WITH DEPC (CD Spectra)

Figure 11. (a) H134C Rieske mutant protein reacted with DEPC over 90 minutes at pH 8.2. (b) Comparison of H134C before and after the DEPC reaction and H134C reacted with a reducing agent. (c) H134C Rieske mutant protein reacted with DEPC over 90 minutes at different pH values. • CD spectra changes are indicative of modification, but not reduction, which

is seen in the truncated wild type protein and many other Rieske mutants. • Reaction rate with DEPC increases with increase in pH.

(a) (b)

H134C REACTION WITH DEPC (UV-Visible Spectra)

Figure 12. Difference spectra of H134C Rieske mutant protein reacted with DEPC over 40 minutes at different pH values. Modification is observed as an increase at 240 nm. As pH increases, an increase in reaction rate can be observed. Inset: Raw data focused on the ligand-to-metal charge-transfer (LMCT) bands. Little change is observed in the LMCT bands indicating no reduction.

(c)

pH-DEPENDENCE OF MODIFICATION

(a) (b)

Figure 13. (a) pH-dependence of modification of H134C with DEPC monitored by UV-Visible spectra (b) monitored by CD spectra. Both graphs show an increase in reaction rate as pH increases.

24 HOUR DEPC REACTION

LMCT Bands of MitoNEET + DEPC (pH = 8.0)

500 1000 1500

0.0

0.2

0.4

0.6

Time (s)

Delta A

bsor

banc

e (A

U) 302 nm

344 nm450 nm

Figure 14. (a) H134C reacting with DEPC over 24 hours observed through the CD at pH 7.6. Changes seen at 442 nm plotted in inset. (b) H134C reacting with DEPC over 24 hours observed using UV Vis at 250 nm, pH 8.2. (c) Truncated wild type Rieske protein reacting with DEPC over 24 hours observed using UV Vis at 250 nm at pH 8.2. (d) MitoNEET reacting with DEPC over time using UV-Vis at various wavelengths.*

* Data collected in the laboratory of Dr. Mary Konkle

(a)(b)

(d)(c)

AKNOWLEDGEMENTS• Dr. Hunsicker-Wang• Past and present members of the Hunsicker-Wang lab• Hart Lab and Alex Taylor at the University of Texas Health Science Center San

Antonio X-Ray core for crystals and data collection• Konkle Group at Eastern Illinois University

1.66 Å resolution

CONCLUSIONS• H134C Rieske mutant appears to be stable at a wide range of pH values. • Titration of H134C using both pH-dependent UV-Visible spectrophotometry and

circular dichroism indicates that the pKo x is 9 . • H134C reacts and becomes modified with DEPC, however reduction is not

observed. • Reaction rate with DEPC is seen to increase with an increase in pH.• Reaction with DEPC monitored overnight shows a reduction in peak that

corresponds with modification, which is similar to what is observed in mitoNEET, and different to the truncated wild type. The decrease may be do to reversal of modification.

FUNDING• Trinity University Department of Chemistry• McNair Scholar Program• National Science Foundation