AbstractThe binding of two fluorinated substrate analogs, 9-fluorocamphor and 5,5-difluorocamphor, to cytochrome P-450-CAM has been investigated by 19F NMR spectroscopy. The NMR properties of each substrate differ depending on whether it is free in aqueous buffer, bound to the diamagnetic ferrous-CO enzyme or bound to the paramagnetic ferrous derivative. As CO must be bound to the ferrous center for it to be diamagnetic, these results demonstrate that camphor and CO bind to the protein simultaneously. The present results are unusual in that the spectral properties of the substrate rather than those of the heme iron have been monitored to follow substrate and ligand binding

Crull, George B.

Dawson, John H.

Nardo, Joseph V.

Elsevier - Publisher Connector 

Volume 254, number 1,2, 39-42 FEB 07537 August 1989 
Direct observation of substrate binding to ferrous-CO cytochrome 
P-450-CAM using lgF NMR 
George B. Crull*, Joseph V. Nardo+ and John H. Dawson 
Department of Chemistry, University of South Carolina, Columbia, SC 29208, USA 
Received 30 May 1989; revised version received 10 July 1989 
The binding of two fluorinated substrate analogs, 9-fluorocamphor and 5,5-difluorocamphor, tocytochrome P-450~CAM 
has been investigated by r9F NMR spectroscopy. The NMR properties of each substrate differ depending on whether 
it is free in aqueous buffer, bound to the diamagnetic ferrous-CO enzyme or bound to the paramagnetic ferrous derivative. 
As CO must be bound to the ferrous center for it to be diamagnetic, these results demonstrate that camphor and CO 
bind to the protein simultaneously. The present results are unusual in that the spectral properties of the substrate rather 
than those of the heme iron have been monitored to follow substrate and ligand binding. 
NMR, t9F-; Cytochrome P-450; Oxygen activation; Monooxygenase; Substrate binding; Ligand binding 
1. INTRODUCTION 
The P-450 cytochromes are a family of heme 
iron enzymes with similar electronic absorption 
properties that activate dioxygen for incorporation 
into unactivated hydrocarbons [l-8]. Addition of 
CO, a potent inhibitor, to the paramagnetic deoxy- 
ferrous enzyme produces the diamagnetic ferrous- 
CO state. This species has a Soret electronic ab- 
sorption band near 450 nm from which the name 
P-450 was derived. The camphor-metabolizing 
cytochrome P-450-CAM from Pseudomonas 
putida, the first P-450 to be purified [2], is soluble 
and has been the frequent choice for physical 
studies of the P-450 system. P-450-CAM stereo- 
and regiospecifically converts (lR)-(+)-camphor to 
5-exohydroxycamphor (scheme 1) [2,4-61. When 
the primary site of hydroxylation is completely 
blocked in 5,5-difluorocamphor, 9-hydroxy- 
Correspondence address: J.H. Dawson, Department of 
Chemistry, University of South Carolina, Columbia, SC 29208, 
USA 
* Present address: Department of Chemistry, State University 
of New York at Stony Brook, Stony Brook, NY 11794, USA 
+ Present address: Cytogen Corp., 201 College Road East, 
Princeton, NJ 08540, USA 
camphor is the product [9]. The stereospecificity of 
P-450~CAM, often absent from mammalian P-450 
[l], coupled with the ability of the enzyme to 
hydroxylate unactivated substrates increase the im- 
portance of understanding the nature of the active 
site. In this regard, Poulos and co-workers [lo-131 
have recently reported X-ray crystal structures of 
ferric P-450-CAM with and without camphor 
bound. 
+ NADH + H+ + 02 QH + NAD+ + H20 
Scheme 1 
The interaction of camphor with ferrous and 
with ferrous-CO P-450-CAM has been investigated 
previously. The Kd value for camphor is identical 
for the two states, suggesting that CO binding does 
not interfere with substrate binding [14]. The af- 
finity of CO for the ferrous enzyme is, however, 
diminished lo-fold when camphor is bound [15]. 
Furthermore, although the Soret electronic absorp- 
tion band of the ferrous-CO enzyme shifts by only 
1 nm upon camphor binding [ 161, significant 
Published by Ekevier Science Publishers B. V. (Biomedical Division) 
00145793/89/$3.50 0 1989 Federation of European Biochemical Societies 39 
Volume 254, number 1,2 FEBS LETTERS August 1989 
changes in the energies of the Fe-C and C-O stret- 
ches of the ferrous-CO enzyme are observed 
[ 17,181. The perturbations in spectroscopic proper- 
ties of the Fe-CO unit caused by camphor binding 
have led to the conclusion that the binding pocket 
of the enzyme can accommodate camphor and CO 
simultaneously. The present study addresses this 
issue from the perspective of the substrate. In par- 
ticular, we have observed significant differences 
with the 19F NMR spectrum of two enzyme-bound 
fluorocamphor substrate analogs (fig.1) in the 
presence and absence of CO. As in the earlier 
work, we conclude that camphor and CO bind to 
the ferrous enzyme concurrently. 
2. MATERIALS AND METHODS 
All commercially available chemicals were of reagent grade 
from Aldrich or Fisher. The syntheses of 9-fluorocamphor and 
5,5difluorocamphor have been reported [9,19]. Cytochrome 
P-450-CAM was isolated from P. putida grown on (lR)-(+)- 
camphor as in [20-221. The concentration of the ferric enzyme 
was determined from the intensity of the Soret band at 391 nm 
[20]. Enzyme purity was established from the 391/280 nm ab- 
sorbance ratio [20-221; samples with ratios above 1.4 have been 
found to be electrophoretically homogeneous and were used in 
this study. Substrate binding was carried out as described [9,22]. 
A Bruker WH-400 NMR spectrometer with a is’F frequency of 
376.13 MHz and controlled by an Aspect 2000A computer was 
employed. All data were obtained using a 10 mm ‘%/‘H dual- 
frequency probe. FIDs were collected using 8K word data tables 
that were zero-filled to 16K words before transforming. The use 
of 15000 Hz sweep width gave digital resolution of 
-0.9 Hz/pt. 
Protein solutions for NMR experiments were concentrated to 
2 mM in 0.1 M potassium phosphate buffer, pH 7.4, with 20% 
DrO as an internal ock. Sample temperature was maintained at 
4°C during handling. Sample volumes of 2.5 ml were used in 
10 mm Wilmad Royal Imperial NMR tubes septum-sealed 
under nitrogen. Ferrous P-450-CAM was prepared in a Vacuum 
Atmospheres dry box by reduction with a 5-fold excess of 
NazS204. The ferrous-CO state was generated by passing a 
stream of CO over the ferrous enzyme. Complete reduction and 
CO coordination were each verified by electronic absorption 
spectroscopy on an aliquot diluted lOO-fold with anaerobic buf- 
fer. Protein integrity was checked by examination of the elec- 
tronic absorption spectrum of the ferrous-CO derivative; essen- 
3 
Fig.1. Structure of the fluorocamphors used in this investiga- 
tion. 
tially no P-420 (inactive P-450 [l-6]) was present at the start and 
less than 5% at the completion of any experiment. 
3. RESULTS AND DISCUSSION 
Binding of camphor to ferric P-450~CAM con- 
verts the heme iron from low- to high-spin. This 
change, easily detectable by the 27 nm blue-shift in 
the Soret absorption band, results from displace- 
ment by camphor of the distal water ligand to the 
heme iron [ 11,131. Camphor binds to a site adja- 
cent to the heme iron but does not coordinate to the 
metal [ 11,131. Upon CO binding to the ferrous en- 
zyme, a spin state change also occurs, from high to 
low, and the Soret band shifts from 408 to 446 nm. 
As discussed above, camphor binding perturbs the 
spectral properties of the ferrous-CO center 
[ 14-181 suggesting that CO and camphor bind to 
the active site simultaneously. Here, we assess this 
issue from the perspective of the substrate in order 
to determine whether CO binding perturbs the 
spectroscopic properties of the substrate. 
The 19F NMR spectrum of 5,5-difluorocamphor 
in aqueous buffer (fig.2A) is an AB pattern com- 
prised of four resonances. The signals at - 10.3 
and - 11.5 ppm have been assigned to the exo 
fluorine and those at - 21.6 and - 22.9 to the endo 
fluorine. Each fluorine produces two signals due to 
scalar coupling to the other fluorine. These 
assignments have been derived from the 19F and ‘H 
NMR spectra of 5,5-difluorocamphor in di-chloro- 
form. In particular, the ‘H NMR resonance of the 
proton at position 4 shows a Jn_r coupling of 
6.8 Hz. Only the 5-exo fluorine has the appropriate 
dihedral angle to produce such coupling. The 19F 
NMR spectrum of 9-fluorocamphor exhibits an 
AXX ’ pattern and has been assigned [ 191. Upon 
binding to ferrous-CO P-450-CAM, new 
resonances appear for 5,5-difluorocamphor 
(fig.2B) and 9-fluorocamphor [19]. The new 
resonances in fig.ZB, summarized along with all 
other NMR data in table 1, correspond to the 
fluorine(s) of the enzyme-bound fluorocamphors. 
The changes in chemical shift of l-2.5 ppm can be 
attributed to a microenvironmental effect upon 
substrate binding to the protein, possibly due to the 
ring current of the heme. 
The NMR properties of the two fluorocamphor 
substrate analogs are again altered upon binding to 
the ferrous enzyme. For 9-fluorocamphor, the 19F 
40 
Volume 254, number 1,2 FEBS LETTERS August 1989 
H 
1000 Hz 
liH 1000 Hz 
l I I I I 
-5 -10 -15 -20 -25 
Shift (ppm) 
Fig.2. (Top) 376 MHz r9F NMR spectrum of 5,5difluoro- 
camphor in potassium phosphate buffer, 20% DzO, pH 6.8. 
(Bottom) 376 MHz 91F NMR spectrum of excess 5.5difluoro- 
camphor bound to 2 mM ferrous-CO P-450-CAM in 100 mM 
potassium phosphate buffer, 20% DzO, pH 6.8. 
resonance shifts another 3.4 ppm from its location 
in the ferrous-CO case (table 1) and is broadened 
lOO-fold as a result of being in close proximity to 
the paramagnetic ferrous iron. Quantification of 
this effect using the Solomon-Bloembergen equa- 
tion [23,24] to determine an average Fe-F distance 
of 3.8 A has been reported [19]. As carbon 5 of 
camphor is closest to the heme iron of P-450-CAM 
[ 11,251, it is perhaps not surprising that no 
resonances are detected for the fluorines of 5,5-di- 
fluorocamphor bound to ferrous P-450-CAM. 
The possibility that the fluorocamphors bind to 
a site other than the active site can be eliminated by 
examination of the data for 9-fluorocamphor- 
bound ferrous P-450-CAM. In the presence of ex- 
cess substrate, two sets of resonances are observed, 
one from camphor free in solution and a second 
from camphor bound within the active site (table 
1). Binding of substrate to an additional site would 
have been expected to produce a third set of 
resonances. The possibility of rapid chemical ex- 
change between bound and free states has been rul- 
ed out both by previous studies [26] and by the ap- 
pearance of separate signals for the bound and free 
fluorocamphors (table 1). If the fluorocamphors 
were in fast exchange, a single resonance would ap- 
pear at a chemical shift corresponding to the mole 
fraction weighted average of the two chemical 
shifts. This is clearly not the case as shown in 
fig.2B. 
In summary, the NMR properties of the two 
fluorocamphor substrate analogs differ depending 
on whether the probe is free in solution, bound to 
the paramagnetic enzyme or bound to the dia- 
magnetic state. Since CO must be coordinated for 
the ferrous iron to be diamagnetic, the substrate 
and CO must be bound to the enzyme at the same 
time. The present results complement earlier 
studies of camphor binding to ferrous-CO 
P-450-CAM in reaching the same conclusion by 
Table 1 
Fluorine chemical shifts 
Camphor Species Chemical shift” 
5,5-Difluoro- free in bufferb - 10.3c, -11.5, -21.6, -22.8 
5,5-Difluoro- bound to diamagnetic ferrous-CO P-450-CAM - ll.lC, -12.3, -20.9, -22.1 
9-Fluoro- free in bufferb - 156.6, - 156.7, - 156.9 
9-Fluoro- bound to diamagnetic ferrous-CO P-450-CAM - 154.ld 
9-Fluoro- bound to paramagnetic ferrous P-450~CAM - 150.7d 
* In ppm relative to external trifluoroacetic acid. Data for 9-fluorocamphor have been previously 
reported [ 191 
b Buffer: 100 mM potassium phosphate, 20% DrO, pH 7.4 
’ The first pair of signals have been assigned to the exo fluorine. Each fluorine gave two 
resonances due to the Jr-r coupling 
d The fluorine-proton coupling, JF.H, was smaller than the observed linewidth 
41 
Volume 254, number 1,2 FEBS LETTERS August 1989 
monitoring the spectral properties of the substrate 
rather than those of the ferrous-CO heme iron. 
Acknowledgements: This work was supported by the National 
Science Foundation (DMB 86:05876 to J.H.D. and CHE 
86: 13421 to the SE Regional NMR Facility). We wish to thank 
Dr Allen .I. Benesi, Helga J. Cohen and Dr A. Ronald Garber 
for their assistance in performing several of these experiments. 
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42 


Direct observation of substrate binding to ferrous-CO cytochrome P-450-CAM using 19F NMR 

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Direct observation of substrate binding to ferrous-CO cytochrome P-450-CAM using 19F NMR

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