SUPPLEMENTARY MATERIAL
Modulation of the reactivity of multiheme cytochromes by site-directed mutagenesis: moving towards the
optimization of microbial electrochemical technologies
Alexandra S. Alves#, Nazua L. Costa#, Ming Tien§, Ricardo O. Louro#, Catarina M. Paquete#*
Table S1. Thermodynamic parameters of STCwt obtained in Fonseca et al. [22] and obtained in
this work by fitting the NMR data of Fonseca et al. together with PFV data. The fully reduced
and protonated state of the protein was taken as the reference state for all hemes. The energies of
oxidation of hemes I, II, III and IV (EI-IV) reported in units of meV correspond to the value of the
reduction potentials of each individual heme according to ∆G=-FE and the pKa derives from the
energy of deprotonation of the ionizable center gH in meV according to pKa=gHF/(2.3RT). The I
values represent the interaction energies between the hemes and between the hemes and the
ionizable center, reported in units of meV. Values in parenthesis represent the standard errors
calculated from the diagonal terms of the covariance matrix considering uncertainty of the NMR
signals proportional to their line width and an uncertainty of 3% in the PFV data. The parameters
related with the redox-Bohr effect were not fitted as explained in the main text.
STCwt
EI
EII
EIII
EIV
II_II
II_III
II-IV
III-III
III_IV
IIII-IV
gH
II-H
III-H
IIII-H
IIV-H
-243
-222
-189
-171
28
21
11
72
11
29
500
-31
-33
-56
-9
8(3)
68(2)
6(2)
25(2)
500
-31
-33
-56
-9
STCwt
-211(3) -190(2) -157(3) -140(4) 23(3) 16(4)
(PFV)
1
Table S2. Fraction of electrons that enters or leaves, through each heme, of STCwt and STC with
his-tag, calculated at pH 7.0 using the kinetic model with the thermodynamic parameters obtained
by fitting the NMR data of Fonseca et al. [22] together with PFV (Table S1 and Table 1).
Fraction of electrons that enters STC
Fraction of electrons that leaves STC
Heme I
Heme II
Heme III
Heme IV
Heme I
Heme II
Heme III
Heme IV
STCwt (PFV)
4.00
0.00
0.00
0.00
3.02
0.00
0.98
0.00
STC His-tag
4.00
0.00
0.00
0.00
3.33
0.01
0.66
0.00
2
Table S3. Macroscopic pKa values for ionizable center associated with the five stages of
oxidation of STC. Values calculated with the thermodynamic parameters of STCwt [22] using
pKa = gH F / (2.3 RT).
STC
Stage 0
Stage 1
Stage 2
Stage 3
Stage 4
8.5
7.9
7.3
7.0
6.3
3
Figure S1. 1H NMR spectra of the misfolded mutants D7N and D81N compared with STCwt.
4
Figure S2. 1H NMR spectra of the STC mutants with the heme geometrical conformation similar to the
STCwt. Some of the heme ring substituents resonances from STC are labeled using the IUPAC-IUB
nomenclature.
5
Figure S3. The pH dependence of the oxidized fraction of heme methyl group resonances 181CH3I,
71CH3II, 21CH3III and 181CH3IV for STC with his-tag and all the STC mutants studied in this work at 25ºC
(stage 3 -
, stage 2 - , stage 1 -
). The lines correspond to the simultaneous fit of the thermodynamic
model to the NMR and PFV data of STC with his-tag for each individual stage of oxidation. The oxidized
fractions in the fully reduced and in the fully oxidized stage are 0 and 1, respectively.
6
Figure S4. Graph of the thermodynamic parameters obtained for STC with his-tag and for each STC
mutant. (A) Energies of oxidation of hemes I, II, III and IV and (B) interactions energies between the
hemes given in Table 1.
7
8
Figure S5. The pH dependence of the oxidized fraction of heme methyl group resonances 181CH3I,
71CH3II, 21CH3III and 181CH3IV for STC with all the mutation at 25ºC (stage 3 -
, stage 2 - , stage 1 -
). The lines correspond to the simultaneous fit of the thermodynamic model to the NMR and PFV data
of each STC for each individual stage of oxidation. The oxidized fractions in the fully reduced and in the
fully oxidized stage are 0 and 1, respectively.
9
Figure S6. Potentiometric titration for all the STC mutants studied in this work obtained by PFV at pH
7.6. The lines correspond to the simultaneous fit of the thermodynamic model to the NMR and PFV data
of each STC for each individual stage.
10
Heme II
Heme III
Heme I
Heme IV
Figure S7. Individual heme oxidation fractions (labeled with Roman numerals) for STC with his-tag. The
dashed line indicates the global oxidation fraction of the protein. The curves were calculated as a function
of the solution reduction potential at pH 7 using the parameters listed in Table 1.
11
12
13
Figure S8. Kinetics of reduction of the STC proteins (STC with his-tag and STC mutants) by sodium
dithionite at different pH values. Gray lines are the kinetic data and black lines are the fit of the kinetic
model to the data.
14
Figure S9. Kinetics of oxidation of the STC proteins (STC with his-tag and STC mutants) by Fe-NTA.
Gray lines are the kinetic data obtained for STC starting from the fully reduced state and from partially
reduced protein and black lines are the fit of the kinetic model to the data.
15