1. Purification of c7 type
cytochromes from
Geobactar sulfurreducens
Argonne National Laboratories
Bioscience Division
Summer 2012
Tom Hajek
2. Overview
What is Geobactar sulfurreducens?
What processes do we use to get the desired
protein?
Why are we so interested in this particular
protein?
Results
Long Term Goals
3. Our Friend
Geobactar sulfurreducens
Found in soil and
sediments.
“Eats” U and other
heavy metals.
Currently being
used in
bio-remediation of
ground water.
Produces MANY
multi-heme
proteins (cytochromes).
4. 4
• Periplasmic cytochrome A (PpcA)
• The most abundant cytochrome in the periplasm of G. sulfurreducens
• Involved in iron (III) reduction and can reduce other metals, e.g. U(VI),
Tc(VII)
Cytochrome c7, PpcA
Londer et al., BBA, 1554 (2002), 202-211
5. What are
Cytochromes?
5
Cytochromes c: cytochromes with covalently bound heme(s)
Electron transfer proteins that carry heme as a prosthetic group
Involved in photosynthesis & aerobic/anaerobic respiration
Function is related to the valence change of heme iron, Fe2+
↔ Fe3+
6. First we must clone....
The gene that
codes for PpcA (c7)
is cloned into E. coli
and selected for by
plating with
antibiotics
Only the cells that
are resistant and
contain our gene of
interest can grow.
7. The lac-operon with c7 gene
(region of interest)
lac promoter OmpA leader c7 gene (mature)
Y. Londer et al 2002
XbaI HindIII
8.
9. Role of IPTGRole of IPTG
RNA polymerase is
“stuck” on promoter
while the repressor
is bound to
operator.of the lac
operon.
IPTG acts as an
analogous substrate
of allolactase and
transcription can
begin.
16. 10 20 30
C7-1 A D D . I V L K A K N G D V K F P H K A H Q K A V P D C K K C H E . K G P G K I
C7-2 A D T . M T F T A K N G N V T F D H K K H Q T I V P D C A V C H G . K T P G K I
C7-3 I D K . I T Y P T R I G A V V F P H K K H Q D A L G E C R G C H E . K G P G R I
C7-4 A D . V I L F P S K N G A V T F T H K R H S E F V R E C R S C H E . K T P G K I
C7-5 H D K V V V L E A K N G N V T F D H K K H A G V K G E C K A C H E T E A G G K I
40 50 60 70
C7-1 E G F G K E M A H G K G C K G C H E E M K K G P T K C G E C H K K - - - - PpcA
C7-2 E G F G K E M A H G K S C K G C H E E M K K G P T K C G E C H K K - - - - PpcB
C7-3 D G F D K V M A H G K G C K G C H E E M K I G P V R C G D C H K G G S T H PpcC
C7-4 R N F G K D Y A H . K T C K G C H E V R G A G P T K C K L C H T G - - - - PpcE
C7-5 A G M G K D W A H . K T C T G C H K E M G K G P T K C G E C H K K - - - - PpcD
Aligned sequences of the homologs illustrating the different distribution of charged residues
(acidic residues Asp and Glu shown in red and basic residues Lys and Arg shown in blue).
Insertions and deletions of residues also result in different arrangements in space of the side
chains causing variation in surface electrostatic potential.
PpcA c7-1 amino acid sequence
K = Lysine E = Glutamic Acid
CxxCH at residues
27-31, 51-55, 65-69
17. E39C, K70C, K29C
71 amino acids.
Contains 3 heme
groups.
“Pocket” in protein
may accept a
photosensitizer.
Cysteine
(Cys)
K = Lysine E = Glutamic Acid
18. 18
Heme I
Heme III
Heme IV
K70
K29
E39
Cysteine Mutation sites in PpcA studied
(E39C, K29C, K70C)
K = Lysine E = Glutamic Acid
22. Challenge: Combine Chemical and Biological Electron
Transfer Function in Metalloprotein Hybrid
22
hv
Catalyst
Photo-
sensitizerLinker:
PhotosensitizerCatalyst
2H+ H2
2e-
Chemical Solar Catalyst Bio-hybrid Solar Catalyst
hv
hv
e-
fastfast
Problem: Efficiency limited by
inability to “re-charge” PS
Solution: Use multi-heme protein
frameworks as source for multiple e-
Artero et. al 2008 Ang. Chem. Int. Ed. 47: 564
X
PI: D. Tiede (CSE-ANL)
