À1
K
m
to 2.0 Æ 0.2 mM and kcat was 34.1 Æ 0.8 s . The NADH
As the ferredoxin potential became less reducing there was a
À1
turnover rate was 19.4 Æ 0.7 s , 12 times that of WT PuxB and
f
corresponding decrease in k , indicating that the thermodynamic
À1
close to the 24.0 s value for Pux. Interestingly, when the F73G
driving force was an important factor in the observed trend in
electron transfer rates and hence kcat. We inferred that the higher
thermodynamic driving force for CYP199A2 reduction by WT
PuxB (ca. 40 mV more reducing than Pux) compensated for the
likely lower donor–acceptor coupling and higher reorganisation
energy for electron transfer arising from sub-optimal residue
interactions. This led to a higher ket and thus kcat for supporting
mutation was introduced to WT PuxB, K was unchanged while
m
kcat was raised slightly. Adding the R29S mutation to the PuxB-5/
E36V/F73G mutant lowered the activity while the M70L
mutation lowered K
À1
m
(1.4 Æ 0.1 mM) but kcat was also lowered
À1
(
27.3 Æ 0.5 s ), leading to a NADH turnover rate of 14.1 s . All
mutants showed the same product selectivity and similar efficiency
of NADH utilisation for product formation as WT Pux and PuxB
m
CYP199A2 turnover that partially overcame the high K ,
(
data not shown).
resulting in a readily detectable activity. Faster electron transfer
from PuxB mutants to CYP199A2 should be possible if different
These results are the first report of ferredoxin engineering to
support native-like P450 activity and provide new insights into
ferredoxin-P450 recognition. The E36V mutation lowered Km
slightly; P450-associated ferredoxins have a hydrophobic residue
at this position close to the [2Fe–2S] cluster while isc ferredoxins
have an acidic residue (Fig. S1, ESIw). The tipping point was the
m
amino acid substitutions can be found that lower K of PuxB-5
while maintaining the ferredoxin potential at more reducing
values. Other isc ferredoxins with lower reduction potentials
6a
fd
6c
than PuxB, e.g. Fdx (À380 mV) and Etp (À353 mV), may
be even better starting points for P450 recognition engineering.
In summary, the results establish, for the first time, the principle
of tailoring a non-physiological ferredoxin to support native-like
P450 activity. The kcat for PuxB reached a native-like value when
residues 42–44 in the cluster binding loop matched those in Pux,
and further increases in turnover activity came from improved
7
-fold lowering of K
m
when the F73G mutation was introduced
to the PuxB-5 mutant. The acidic residue at the 72 position in
2,7
the a3 helix is important in P450 binding, and it is not
surprising that the side chain volume at residue-73 has such an
impact. An important observation is that the F73G mutation
was only effective when combined with the mutations in the
PuxB-5 mutant. Overall the data showed that ferredoxin–
CYP199A2 binding requires collective residue matches at 36,
m
protein binding to lower the K . Since the structures of isc
and P450-associated ferredoxins are closely similar in the P450
recognition region, it should be possible to tailor other ferredoxins
to reconstitute the activity of CYP199A2 and potentially other
known and new P450 enzymes.
3
8, and 105 on one side of residues 42–44 in the cluster binding
loop and residues 66, 69, 72 and 73 on the other side. Interestingly
the effects of the R29S and M70L mutations indicate that perfect
residue matches with the physiological ferredoxin is not required
for activities that are useful in practice.
We thank the EPSRC, BBSRC, the Rhodes Trust and
NSERC (Canada) for financial and studentship support of
this work.
Intra-complex electron transfer from Pux and the most
active PuxB mutants to CYP199A2 was studied by stopped-
flow spectrophotometry. Pre-reduced ferredoxins were mixed
with oxidised CYP199A2 in the presence of a saturating
concentration of 4-methoxybenzoic acid under an atmosphere
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6
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1694 Chem. Commun., 2012, 48, 11692–11694
This journal is c The Royal Society of Chemistry 2012