Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and
Cytochrome b5 Observed by Solid-State NMR Spectroscopy
Kazutoshi Yamamoto,1 Ulrich H. N. Dürr,1 Jiadi Xu,1 Sang-Choul Im,2 Lucy Waskell,2 and
Ayyalusamy Ramamoorthy1*
1
Biophysics Research Division and Department of Chemistry, University of Michigan, Ann
Arbor, MI 48109-1055
2
Department of Anesthesiology, University of Michigan, and VA Medical Center, Ann Arbor, MI
48105
Supporting information
Amino acid sequence of rabbit cytochrome b5, the transmembrane domain is
underlined.
MAAQSDKDVK10-YYTLEEIKKH20-NHSKSTWLIL30 -HHKVYDLTKF40 LEEHPGGEEV50 -LREQAGGDAT60 -ENFEDVGHST70-DARELSKTFI80IGELHPDDRS90-KLSKPMETLI100-TTVDSNSSWW110-TNWVIPAISA120LIVALMYRLY130-MADD
Expression of 15N labeled full-length rabbit cytochrome b5 and wild type
cytochrome P450 2B4
15
N labeled full length rabbit cytochrome b5 was overexpressed in E.coli C41 cells using
a pLW01 plasmid.1 After adaptation of the cells in 15N-enriched Celtone medium
(Spectra stable Isotopes), the cells were transferred into a large volume of the 15NCeltone culture (called Celtone-N). The 15N-Celtone culture contained 880 mL of
Celtone-N solution, 112 mL of M9 solution, 2 mL of glycerol, 2g of [ 15N]ammonium
sulfate, 0.24 mM carbenicillin, 1 mM CaCl2, 10 mM MgSO4, 10 M FeCl3, 25 M
ZnSO4, 20 M MnCl2, 250 M -aminolaevulinic acid and vitamin mix (25 g of biotin,
choline chloride, folic acid, niacinamide, D-panthenate, pyridoxal-HCl, riboflavin and
250 g of thiamine). Optimal expression was achieved with a 100 mL culture volume in
S1
a 500 mL Erlenmeyer flask. The flask was incubated at 35 oC and spun at 250 rpm until
OD at 600 nm reached 1.2. Isoprppyl -D-thiogalactopyranoside (IPTG) was added to
the final concentration of 10 M, and incubation was continued for 20 h at which the
cells were harvested. Purification of the 15N cytochrome b5 (cyt b5) was performed as
described elsewhere.2 Expression and purification of the cytochrome P450 2B4 (cyt
P450) were performed as described in the literature.1, 3
15% SDS PAGE gel of 15N labeled full length rabbit cytochrome b5
Purification has resulted in highly purity samples on SDS PAGE gels. 4 g of purified
15
N cyt b5 was loaded onto 15% SDS PAGE gel and ran for 50 minutes at 150V. Soluble
(no tail) bovine b5 was used for comparison to 15N labeled cyt b5. Results show that the
15
N labeled b5 shows a single band at 18 kDa and 15N labeled rabbit b5 still has an anchor
region after several weeks of solid-state NMR experiments. Sine solid-state NMR
measurements utilizes high radiofrequency power and cyt-b5 is heat sensitive, we
regularly confirmed the stability of the protein using this procedure.
Soluble b5 (bovine b5)
15
N rabbit b5 (before ssNMR)
15
N rabbit b5 (after ssNMR)
marker
21K
14K
Figure S1. 15% SDS PAGE gel of cyt b5 showing that 15N labeled b5 is a single band at
18 kDa and 15N labeled b5 still has an anchor after several weeks of solid-state NMR
experiments.
The electron transfer scheme for the enzymatic activity of cyt P450.
S2
Figure S2. Electron transfer scheme and the molecular models of cyt b5, cyt P450 and
cyt b5-cyt P450 complex. The reaction catalyzed by an individual cyt P450 is the
oxidation of the substrate (denoted as ‘X’ in the scheme). The transmembrane region of
cyt b5 has been shown to be essential for to enable its interaction with and activation of
cyt P450.4-6
Assay of cyt b5-cyt P450 complex
Cytochrome b5 acts as a second electron donor for cyt P450 metabolism. Experimental
measurements of the change in the spin state, double mutant cycle analysis, and kinetic
data, it was confirmed that cyt b5 forms a complex with cyt P450. The cyt b5 binding
changes environment of a substrate in the active center of cyt P450 and then alters the
spin state of the cyt P450 heme iron from a low-spin hexacoordinate heme to high-spin
pentacoordinate heme, which is called type I change. Dissociation constant, Kd, of cyt
b5-cytP450 complex was determined by measuring type I spectral change occurring when
cyt b5 was added to a cyt P450 sample containing substrates such as benzphetamine (BZ)
or methoxyflurane (MF) and phosphatidylcholine (DLPC). Absorbance measurements
were performed on a Cary-3 (Varian) dual beam spectrophotometer. The sample cuvette
contained 1 mL of 50 % MF saturated 100 mM KP buffer, pH 7.4, 20 % glycerol, 0.3 M
cyt P450 2B4 and 24 M DLPC. The reference cuvette contained the same mixture as
the sample cuvette except cyt P450. The cuvettes were incubated for five minutes at 25
o
C and then recorded the base line using the dual beam in the spectrophotometer. Cyt b5
(0.04-2 nmol) was added to each cuvette and then mixed gently. After 2 minutes, total
absorbance changes of type I (between 420 and 385 nm) spectra were recorded (see
Fig.1C in the main text). Using the absorbance changes and concentration of cyt b5, the
Kd value was determined by using the following equation,
é (K + [P ]+ [B ])
0
0
DA = Amax ê d
2
êë
(K d + [P0 ]+ [B0 ])2
4
ù
- [P0 ][B0 ]ú ,
úû
where [P0] is the initial concentration of P450 and [B0] is the initial concentration of cyt
b5, the independent variable. Kd and Amax were the constants and determined using a
SigmaPlot software.
Preparation of Bicelles
40 mg of DMPC and 7.6 mg of DHPC corresponding to a molar ratio of
q=[DMPC]/[DHPC]=3.5 were cosolubilized in chloroform. Solvent was removed under
a stream of N2 gas to produce a lipid film on the walls of a glass tube, which was kept in
vacuum overnight to remove all residual solvents. 65 l of 50 mM HEPES buffer, pH
7.4, with 10% glycerol content was added to the lipid film and transferred to a 5 mm
NMR tube for experimental measurements. The resulting mixture of extreme viscosity
was homogenized by vortexing, 30 min gentle sonication in an ice bath, and 4 freeze/heat
cycles between liquid nitrogen and 50 °C. The resulting turbid gel is still extremely
viscous, but its viscosity was slightly reduced at 0 °C, as is common in all bicellar
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preparations. Protein was added in the final step of the preparation of biclles. Adding 55
l of 2.0 mM cyt b5 (or cyt b5-cytP450 complex) solution corresponding to 1.76 mg or
110 nmol protein resulted in a protein:DMPC ratio of 1:536. 35 l of 50 mM HEPES
buffer, pH 7.4, with 10% glycerol content was further added to the sample. Once the
components were mixed, the sample tube was sealed and the sample was placed inside
the NMR probe for subsequent NMR experiments. The degree of sample alignment was
measured using 31P-NMR and was found to be stable after 1 hr of equilibration.
Best fit details
The best-fit helical wheel patterns was analyzed and obtained by using a home-written
program. First, the positions of all the clearly resolved peaks were extracted as pairs of
15
N chemical shift and 1H-15N dipolar coupling values. Then, these pairs were arranged
to fit onto the PISA wheels as predicted by geometrical calculations. The equation used
in these calculations can be found in the literature. The best-fit helix tilt for cyt b5 with
bicelles was found to be 14.0° at a molecular order parameter of Smol= 0.70, and for cyt
b5-cyt P450 complex was 16.0° at a molecular order parameter of Smol= 0.76. The error
of fit in PISA wheels at slightly varying tilt angles was calculated to be ±3°. More
information on the the tilt angle measurement and error can be found in a previous
publication from our lab.7,8
References
1. Bridges, A. et al. Identification of the binding site o cytochrome P450 2B4 for
cyrochrome b5 and cytochrome P450 reductase. J. Biol. Chem. 273, 17036-17049 (1998).
2. Mulrooney, S. B. & Waskell, L. High-level expression in Escherichia coli and
purification of the membrane-bound form of cytochrome b5. Protein Expr. Purif. 19,
173-178 (2000).
3. Saribas, A. S., Gruenke, L. & Waskell, L. Overexpression and purification of the
membrane-bound cytochrome P450 2B4. Proteins Expr. Purif. 21, 303-309 (2001).
4. Vergéres, G. & Waskell, L. Cytochrome b5, its functions, structure and membrane
topology. Biochimie. 77, 604-620 (1995).
5. Schenkman J. B. & Jansson, I. The many roles of cytochrome b5. Pharmacol Ther. 97,
139-152 (2003).
6. Clarke, T. A., Im, S. C., Bidwai, A. & Waskell, L. The role of the length and sequence
of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis. J.
Biol. Chem. 279, 36809-36818 (2004).
7. Dürr, U. H., Yamamoto, K., Im, S. C., Waskell, L. & Ramamoorthy, A. Solid-state
NMR reveals structural and dynamical properties of a membrane-anchored electroncarrier protein, cytochrome b5. J. Am. Chem. Soc. 129, 6670-6671 (2007).
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8. Ramamoorthy, A., Kandasamy, S. K., Lee, D. K., Kidambi, S. & Larson, R. G.
Structure, Topology and tilt of cell-signaling peptides containing nuclear localization
sequences in membrane bilayers determined by solid-state NMR and molecular dynamics
simulation studies. Biochemistry 46, 965-975 (2007).
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