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INTERMEDIATES IN THE REACTION CYCLE OF
CYTOCHROME P450cam
1,000
0,996
starting material
1,003
relative Transm ission
1,002
Fe(IV)
1,001
1,000
0,999
8ms reaction time
1,000
0,999
0,998
5 min reaction time
-10 -8
-6
-4
-2
0
2
4 -1 6
velocity [mms ]
8
10
V. Schünemann, Technische Universität Kaiserslautern, Germany
C. Jung, KKS Ultraschall AG, Switzerland
A.X. Trautwein, Universität zu Lübeck, Germany
Motivation
The physiologically important enzyme superfamily cytochrome P450 catalyzes a variety
of reactions, such as aliphatic and aromatic hydroxylations, epoxidations, heteroatom
oxidation, and N- and O-dealkylation, by transfer of an active oxygen atom from its
heme unit to the substrates. All enzymes of the cytochrome P450 family have a
hydrophobic binding pocket, in which resides a protoporphyrine IX with its iron center
being coordinated to an axial cysteine ligand [1].
The enzyme cytochrome P450cam from Pseudomonas putida hydroxylases (1R)camphor as natural substrate and is regarded to be a representative enzyme for the
whole P450 family. Mössbauer spectroscopic studies on this enzyme have been
performed by Debrunner and coworkers in the seventies [2] of the last century. The
results of these studies provided insight into the catalytic mechanism of the enzyme
even before the crystal structure of the resting state of P450cam was published [3].
Fig. 1 Cytochrome P450cam
(left) adds an oxygen atom O at
the 5-exo position of camphor
(C10H16O, right).
HO
The cytochrome P450 reaction cycle
The postulated enzymatic reaction cycle is shown in Fig. 2. In the resting state of the enzyme the
catalytically active heme iron center acquires the ferric low-spin state (S=1/2). After binding of the
substrate camphor to the amino acid residue Tyr96 inside the heme pocket, the iron changes from
the ferric low-spin to the ferric high-spin state (S=5/2). The transfer of the first electron originating
from NAD(P)H via redox proteins (flavin and iron-sulfur proteins) reduces the iron to the ferrous
high-spin state (S=2).
Subsequent binding of molecular
oxygen to the iron forms a diamagnetic
FeO2 center, similar to the oxygenated
state of myoglobin. The transfer of a
second electron initiates catalytic steps
which lead to an iron-oxo intermediate
called compound I (cpd I). It is this
intermediate which inserts the active
oxygen atom into the substrate
camphor.
Fig. 2 The reaction cycle of cytochrome
P450. The “shunt“ reaction used in this
study is depicted in red.
The nature of the intermediate cpd I
Neither the electronic structure of cpd I of P450cam nor of any other P450 has been
unambiguously determined up to now, but it is generally assumed that the putative cpd I in
cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I
of peroxidases [4], catalases and many synthetic cpd I analogues [5]. In these systems one
oxidation equivalent resides on the Fe(IV)=O unit (d4, S=1) and one is located on the porphyrin
(S'=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin -cation radical system.
For other enzymes like cytochrome c peroxidase a ferryl Fe(IV) coupled to an organic radical on
the protein (SRadical=1/2) has been proposed [6].
R-H + O2 + 2e- + 2H+ 
S Fe(IV)=1
R-OH + H2O
S Radical=1/2
Fig. 3 The Intermediate cpd I (left) inserts oxygen into the substrate R-H via the reaction
shown on the right.
Experimental techniques
Reaction intermediates can be trapped by rapid freeze-quench experiments (Fig. 4) . This
technique allows to prepare samples for Mössbauer- and electron paramagnetic resonance
(EPR-) spectroscopy. In order to detect cpd I in cytochrome P450cam we have used the “shunt”
reaction (see Fig. 2) with peroxy acetic acid.
•Addition of peroxy acetic acid
to native Cyt P450cam
•Freezing of the reaction mixture
within milliseconds
Investigation with
•Mössbauer Spectroscopy
•Multi-Frequency EPR
Spectroscopy
Fig. 4
The start: Mössbauer Spectroscopy on camphor-free cytochrome P450cam :
The Mössbauer spectrum obtained at 4.2K and B=20 mT of cytochrome P450cam without the
substrate camphor is shown in Fig. 5. The observed magnetic splitting and the corresponding
EPR spectrum (Fig. 6) are consistent with a ferric low-spin state of the iron. The Mössbauer
spectrum has been analyzed by the spin-Hamiltonian formalism (solid line in Fig. 5) and the
parameters are shown in Tab. 1 [7].
g-factors
1,002
2,45
2,2
1,95
1,000
d  ''/dB
relative Transmission
3,2 2,95 2,7
0,998
0,00
0,996
T=4.2K
B=20m T I 
200
300
400
0,994
Fig. 6
-10
Fig. 5
-5
0
5
B [mT]
10
-1
velocity [mms ]
S
1/2

(mm/s)
EQ
(mm/s)
0.38
2.85

-1.8
g
A/gNN
(T)
(1.91,2.26,2.45)
(-45,10,19)
Table 1. Mössbauer parameters used for the spin Hamiltonian simulations of the ferric
low-spin species of cytochrome P450cam (solid lines in Figs. 5 and 7).
Freeze-quench experiments I: 57Fe(III)P450cam + substrate camphor + peroxy acetic acid
The Mössbauer spectrum obtained at 4.2K and B=20 mT of cytochrome P450cam with the
substrate camphor after reaction with peroxy acetic acid for 8 ms is shown in Fig. 7. The spectrum
displays an S=5/2-1/2 equilibrium which is characteristic for the substrate bound P450cam, but no
Fe(IV) has been detected. The corresponding EPR spectrum (Fig. 8) also shows an S=5/2-1/2
equilibrium. No characteristic signals for cpd I with S=1/2 or S=3/2 have been observed, only very
small amounts of an organic radical (0.6%). Obviously the addition of peroxy acetic acid does not
significantly affect the Mössbauer- and EPR pattern of the substrate bound cytochrome P450cam.
1210 8
6
g-factors
4
2
1,003
S =5/2 46%
0,08
S =1/2 23%
1,002
S =1/2 29%
0,06
R adical 0.6%
Fe(III) low -spin, 50%
1,001
d  ''/dB
relative Transmission
Fe(III) high-spin, 50%
1,000
0,999
0,04
S =1/2 1.3%
0,02
0,00
T=4.2K
B=20mT I 
-0,02
0,998
-8
-6
-4
-2
0
2
4
6
8
100
Fig. 8
-1
Fig. 7
velocity [mms ]
S

(mm/s)
EQ
(mm/s)
5/2
0.44
0.79

0.6
200
300
400
B [mT]
g
A/gNN
(T)
D/kB
(K)
E/D
(2.0,2.0,2.0)
(-18,-18,-18)
5
0.087
Table 2. Mössbauer parameters used for the spin Hamiltonian simulation of the ferric –
high-spin species of cytochrome P450cam shown in Fig. 7 (taken from [8]).
Freeze-quench experiments II: Substrate-free 57Fe(III)P450cam + peroxy acetic acid
The Mössbauer spectrum obtained at 4.2K and B=20 mT of substrate-free P450cam after reaction
with peroxy acetic acid for 8 ms is shown in Fig. 9. In addition to the ferric-low spin form (black solid
line with parameters given in Table 1) the spectrum displays a doublet (red line, 132%) with =0.13
mms-1 and EQ=1.94 mms-1 characteristic for a ferryl species ((Fe(IV)=O)2+ ; S=1) [9] which has been
shown to be protonated [10]. The pattern of the starting material is restored after 5 min reaction time.
This shows that the ferryl species is indeed a transient intermediate.
Fig. 9
Fig. 10
3,2 2,7
1,000
The corresponding EPR
spectrum (Fig. 10) shows
an intense radical signal
with a spectroscopic
signature pointing towards
a tyrosyl radical.
g-factors
2,2
0,06
0,996
starting material
0,01
d  ''/dB
1,003
Fe(IV)
1,001
-0,04
0,2
0,0
1,000
-0,2
0,999
0,008
8ms reaction time
200
1,000
300
B [mT]
400
0,004
d  ''/dB
relative Transm ission
1,002
0,000
-0,004
0,999
-0,008
0,998
2,04
5 min reaction time
-10 -8
-6
-4
-2
0
2,02
2,00
1,98
g-factor
2
4 -1 6
velocity [mms ]
8
10
Tyrosyl radical?
1,96
Assignment of the radical signal to Tyr96
A multi-frequency EPR study of the freeze-quenched intermediate obtained from reaction of
substrate-free cytochrome P450cam and its Y96F and Y96F-Y75F mutants with peroxy acids
has been performed [11]. High-field EPR studies at 285 and 94 GHz on freeze-quenched wild
type and Y96F samples reveal g-tensor components for the radical (stretched gx-values from
2.0078-2.0064, gy = 2.0043, and gz = 2.0022), which are fingerprints for tyrosine radicals.
From the simulation of the hyperfine structure in the 94 GHz EPR spectra the signals have
been assigned to Y96 in the wild type and to Y75 in the Y96F mutant.
We suggest that a transiently formed
Fe(IV)=O porphyrin--cation radical
intermediate in P450cam is reduced
by intramolecular electron transfer
from these tyrosines within 8 ms (Fig.
11). Such an intramolecular electron
transfer may be a
general
phenomenon in cpd I formation
(Fig.12).
Fig. 12
Fig. 11
2-
Cytochrome P450cam
substrate-bound
Summary
A complementary Mössbauer- and EPR-study of the time dependence of the reaction of
substrate free P450cam with peracetic acid within a time region ranging from 8 ms up to
5 min has been presented. An Fe(IV) species as well as a tyrosyl radical residing on the
amino acid residue Tyr96 have been identified as reaction intermediates. These species
possibly are formed by the reduction of compound I by means of transferring an electron
from Tyr 96 to the heme moiety.
Readers who are interested in the field of reaction intermediates of cytochrome P450
are referred to a review article by Jung et al. [12].
References
[1] D.F.V. Lewis, Cytochromes P450- Structure, Function and Mechanism, Taylor & Francis Ltd, London,
1996.
[2] M. Sharrock, P.G. Debrunner, C. Schulz, J.D. Lipscomb, V. Marshall and I.C.Gunsalus, Biochim. Biophys.
Acta 420, 8 (1976)
[3] T.L. Poulos, B.C. Finzel and A.J. Howard, Biochemistry 25, 5314 (1986).
[4] R. Rutter, L.P. Hager, H. Dhonau, M. Hendrich, M. Valentine and P. Debrunner, Biochemistry 23, 6809
(1984)
[5] E. Bill, Hyp. Int. 90, 143 (1994)
[6] G. Lang, K. Spartalian and T. Yonetani, Biochim. Biophys. Acta 451, 250 (1976)
[7] M. Sharrock, P.G. Debrunner, C. Schulz, J.D. Lipscomb, V. Marshall and I.C. Gunsalus Biochim. Biophys.
Acta 420, 8 (1976)
[8] V. Schünemann, C. Jung, J. Terner, A.X. Trautwein and R. Weiss, J. Inorg. Biochem. 91, 596 (2002)
[9] V. Schünemann, C. Jung, A.X. Trautwein, D. Mandon and R. Weiss, FEBS Lett. 479, 149
(2000)
[10] R.K. Behan, L.M. Hoffart, K.L. Stone, C. Krebs and M.T. Green, J. Am. Chem. Soc. 128(35), 11471
(2006)
[11] V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.-L. Barra, S.G. Sligar and A.X. Trautwein, J. Biol.
Chem. 279 No.12, 10919 (2004)
[12] C. Jung, V. Schünemann, F. Lendzian, A. X. Trautwein, J. Contzen, M. Galander, L. H. Böttger, M.
Richter and A.-L. Barra, Biol. Chem. 386 No. 10, 1043 (2005)