©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Conserved Glu at the Cytochrome P450 1A2 Distal Site Is Crucial in the Nitric Oxide Complex Stability (*)

(Received for publication, December 29, 1995; and in revised form, January 26, 1996)

Ryosuke Nakano Hideaki Sato Akira Watanabe Osamu Ito Toru Shimizu (§)

From the Institute for Chemical Reaction Science, Tohoku University, Sendai 980-77, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). Both NOS and P450 form stable nitric oxide (NO)-ferric heme complexes, whereas an NO-ferric heme complex of methemoglobin, that has an imidazole-coordinated heme active site, is easily reduced. The NO complex stability of the thiolate-coordinated hemoproteins, however, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate. In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. We found that a mutation at Glu-318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O(2) activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex. Addition of 1,2:3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. Based on these results, together with other spectral and kinetic data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS, is largely ascribed to an ionic bridge between NO and the distal carboxyl group.


INTRODUCTION

The microsomal P450 (^1)class of hemoproteins catalyze monooxidation reactions of a variety of external and endogenous organic compounds with O(2) and electrons ( (1, 2, 3) and references therein). Likewise, NOS produces physiologically important NO from L-Arg and N^G-hydroxy-L-Arg in the presence of O(2) and NADPH ( (4, 5, 6, 7) and references therein). A hemoprotein domain of NOS has a monooxidation catalytic site that is likely to have a thiolate-coordinated heme iron similar to that of P450(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .

NO is a product of the NOS reaction, but is believed to tightly bind to the heme iron(10, 12, 19) . The NO-ferric NOS complex is stable in the absence of O(2), and is similar to P450(10, 12, 19, 20, 21, 22) . On the other hand, an imidazole-coordinated hemoprotein, metHb, is easily reduced by the binding of NO under anaerobic conditions(23) . NO-ferric-porphyrin complexes are also easily reduced in polar solvents (24) . The NO complex stability of the thiolate-coordinated ferric hemoproteins, thus, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate.

Spectral studies of the NO-ferric hemoproteins or water-soluble ferric heme complexes have to date been extremely difficult because of their intrinsic instability toward autoreduction and of NO instability under aerobic conditions(20, 23, 24) . Thus, most of the NO binding studies to hemoproteins have been done for ferrous complexes under anaerobic conditions ( (25, 26, 27, 28, 29) and references therein). Recently, it became feasible to carry out NO-binding studies for ferric hemoproteins as well as ferrous hemoproteins, because of the current commercial availability of NO-releasing (donating) reagents such as NOR1. With the use of this NO donor, spectral titration experiments of NO and laser flash photolysis experiments for ferric P450 enzyme are possible.

In order to understand the interaction between NO and the P450 heme active site, we studied the NO binding to the wild-type and putative distal mutant P450 1A2 enzymes by taking advantage of the NO donor. We found that E318A mutation at the putative distal site of P450 1A2 markedly facilitates the iron reduction with NO. The reducing capability of NO in the E318A mutant was largely influenced by substrates. We discuss the Fe-N-O conformation in the P450 1A2 distal site in association with the NOS distal structure.


EXPERIMENTAL PROCEDURES

Materials

Site-directed mutagenesis, DNA sequencing, expression of wild-type and mutant P450 1A2 proteins in yeast (Saccharomyces cerevisiae), and subsequent purification of expressed P450 1A2 proteins were carried out as described previously (30, 31, 32) . The amino acid sequence at the N-terminal end of the purified expressed P450 1A2 was exactly the same as that of the major form of P450 in methylcholanthrene-induced rat liver microsomes(33) . Purified enzyme was prepared in 0.1 M potassium phosphate buffer (pH 7.4) containing 20% (v/v) glycerol, 1 mM EDTA, and 1 mM dithiothreitol. Concentration of P450 1A2 enzyme was determined from a molar absorption coefficient of 9.3 times 10^4M cm at 447 nm for the [CO-reduced] - [reduced] difference spectrum(30) .

NO was made from NOR1 purchased from Dojindo Laboratories (Kumamoto, Japan). Careful experiments with NOR1 indicated that almost the same (experimental errors within 5%) concentration of NO is released from the equimolar NOR1 in 30 min after NOR1 is mixed with the buffer (pH 7.4) at 25 °C. It was also found the hexeneamide compound derived from NOR1 after the NO release does not bind to the heme iron of the wild-type and mutant P450 1A2 enzymes in terms of the Soret and visible absorption spectra. Namely, addition of a large excess of NOR1 up to 2.0 mM to the P450 1A2 solution did not alter the high-spin band to the nitrogen- or oxygen-coordinated low-spin band under aerobic conditions(30) . Thus the hexeneamide part of NOR1 should not disturb the NO binding to the P450 1A2 heme active site.

7-Ethoxycoumarin, anthracene, phenanthrene, and dibenzanthracene were obtained from Aldrich. All other chemicals of the highest guaranteed grade available were purchased from Wako Pure Chemical Industry (Osaka, Japan) and were used without further purification.

Methods

Optical absorption spectra were obtained at 25 °C with a Shimadzu Model UV-2200 UV spectrometer. NO binding studies were conducted in a gas-tight capped cell (10 times 2 mm) under argon atmosphere. This cell contained an oxygen-free buffer composed of 1.0 µM P450 1A2, 0.1 M potassium phosphate (pH 7.4), 20% (v/v) glycerol, 1.0 mM EDTA, 75 mM glucose, 10 units catalase, and 0.8 units of glucose oxidase. NO titration experiments were repeated at least 3 times for each complex. Regression analyses were performed and lines giving an optimum correlation coefficient were selected. Linear least-square fittings were carried out on a Power Macintosh 6100/60AV personal computer using DeltaGraph software as described previously(33) . Experimental errors were within 25%. For optical absorption and laser flash photolysis studies, we always kept each solution at least 30 min at 25 °C to reach equilibrium after saturated amounts of polycyclic hydrocarbons were added to the solution (30, 31, 32) .

Laser flash photolysis experiments were carried out in the 10 times 1-mm cell at 25 °C with the second harmonic (532 nm) of a Nd:YAG laser (Quanta-Ray, GCR-130) producing an excitation flash of 125 millijoules with a pulse width of 6 ns. Details of this machine are described elsewhere(34, 35) . Kinetic data was analyzed on a Power Macintosh 6100/60AV personal computer using DeltaGraph software. Experimental errors were within 20%.


RESULTS

Interaction of NO with P450 1A2 Enzymes: Optical Absorption Spectral Studies

Addition of NO to ferric wild-type P450 1A2 changed the high-spin form having Soret absorption at 393 nm to an NO-bound low-spin complex having Soret absorption at 431 nm with an isosbestic point at 414 nm (Fig. 1, A and B). The position of the absorption peak of the NO-wild-type P450 1A2 complex was almost the same as those reported for other NO-ferric P450 complexes (Table 1). Double reciprocal plots of the spectral change versus NO concentration formed a straight line, suggesting that 1:1 NO-P450 1A2 complex is formed (Fig. 1C). Spectral K(d) value of NO from the wild-type P450 1A2 was 9.2 µM (Table 2). The K(d) value markedly decreased down to less than 0.25 µM in the presence of anthracene (Table 2). 7-Ethoxycoumarin also decreased the K(d) value (Table 2). In contrast, addition of phenanthrene and dibenzanthracene to the wild-type P450 1A2 increased the K(d) value by 8-fold. L-Arg and N^G-hydroxy-L-Arg did not essentially alter the K(d) value of NO for the wild-type P450 1A2 (Table 2).


Figure 1: Optical absorption spectra of Fe and NO-Fe wild-type P450 1A2 complexes (A), Soret absorption spectral changes of Fe wild-type P450 1A2 caused by the addition of NO (B), and double-reciprocal plots of the absorbance change at 431 nm versus the concentration of free NO in the presence of 7-ethoxycoumarin (0.3 mM) (R^2 = 0.755) (circle) and dibenzanthracene (0.02 mM) (R^2 = 0.885) (bullet) (C). NO titration experiments were repeated 3 times and linear least-square fittings were performed on a Power Macintosh 6100/60AV personal computer with DeltaGraph software as described previously(33) .







Glu-318 in P450 1A2 is conserved as Glu/Asp throughout most P450s and will be important in activating O(2) for catalytic function of P450s(36, 37, 38) . The conserved Glu/Asp is located at the distal site in the heme active site cavity based on the crystal structure of P450cam(39) , P450BM3(40) , P450terp(41) , and P450eryF(42) . An E318D mutation at the putative distal site of P450 1A2 did not change the high-spin type spectrum, but increased the K(d) values of NO by 1.8-fold (Table 2). All substrates except for L-Arg and N^G-hydroxy-L-Arg largely increased the K(d) values for this mutant (Table 2). Especially dibenzanthracene increased 4.7-fold the K(d) values of NO for the E318D mutant. In the presence of L-Arg and N^G-hydroxy-L-Arg, spectral changes of the mutant with NO binding were not linearly increased.

An E318A mutant at the putative distal site of P450 1A2 without NO was almost in the low-spin state(30, 37) . By adding NO to this mutant, a peak at 431 nm ascribed to the NO-ferric low-spin complex was formed. But later, a new peak at 400 nm appeared concomitant with the disappearance of the 431 nm peak (Fig. 2). This new peak is ascribed to a 5-coordinated NO-ferrous complex(20, 21, 22) . The spectral change rate from the NO-bound ferric low-spin complex to the NO-bound ferrous complex of the mutant was 1.2 times 10 min (Table 3) in terms of the spectral change monitored at 431 nm. Interestingly, in the presence of dibenzanthracene, the spin state of the mutant was a high-spin state and essentially no redox change from ferric to ferrous state was observed (Table 3). Phenanthrene also markedly disturbed the redox change by 10-fold. On the other hand, the presence of other substrates did not essentially alter the spectral change rate from NO-bound ferric to ferrous complex (Table 3).


Figure 2: Absorption spectral changes of the E318A mutant (2.0 µM) by adding 0.1 mM NO (A) and time course curves monitored at 431 nm (circle) ascribed to NO-Fe and at 400 nm (bullet) ascribed to NO-Fe after the addition of NO (B).





Note that the 5-coordinated NO-ferrous complex with the 400-nm peak is not denatured. When CO gas was bubbled to the NO-ferrous wild-type complex with the 400-nm peak, only a 450-nm peak appeared, but no peak around 420 nm appeared (Fig. 3). When CO gas was bubbled to the NO-ferrous E318A mutant complex with the 400 nm peak, almost no spectral change was observed, probably because NO affinity to the ferrous mutant will be much higher than CO affinity.


Figure 3: Optical absorption spectra of Fe, dithionite-reduced Fe, NO-Fe, and CO-Fe complexes of the wild-type P450 1A2. The NO-Fe complex was formed by adding NO to the dithionite-reduced Fe complex. The CO-Fe complex was formed by bubbling CO for 2 min to the NO-Fe complex. Due to high affinity of NO to Fe complex, fully CO-bound Fe complex was not formed but apparently mixed with NO-Fe complex in terms of the Soret intensity. Denatured P420 with absorption peak at 420 nm was less than 10% estimated from the absorption intensity.



Thr-319 of P450 1A2 is also very conserved at the putative distal site of P450s and is known to be involved in the O(2) activation associated with the catalytic function of P450s(43, 44, 45) . The K(d) value of an NO-ferric T319A mutant complex was 28 µM, and this is higher than the corresponding values of the wild-type and the E318D mutant (Table 2). However, in contrast to the E318A mutant, no change of the redox state from ferric to ferrous low-spin complex was observed in the NO binding to the T319A mutant.

Geminate Recombination Kinetics of NO to P450 1A2 Enzymes: Laser Flash Photolysis Studies

Geminate recombination rate constants of NO to ferric P450 1A2 enzymes were obtained with laser flash photolysis (Fig. 4). Approximately 1% of the NO molecule coordinated to the wild-type ferric P450 1A2 was dissociated with laser light with power 125 millijoules at 532 nm. Recombination k of NO to the wild-type was 1.7 times 10^4M s (Table 2). The k value was increased up to 11-fold in the presence of 7-ethoxycoumarin. Anthracene, phenanthrene, and dibenzanthracene also raised the k value more than 2-fold up to 8-fold (Table 2). In the presence of L-Arg and N^G-hydroxy-L-Arg, NO binding pattern was not monotonous, thus it was not feasible to obtain the geminate recombination rate constant of NO to the wild-type P450 1A2.


Figure 4: Laser flash photolysis of the NO-wild type complex. A, laser flash photolysis time course curve at 431 nm for the NO rebinding to the Fe wild-type (8 µM) in the presence of 2 mM NO. Other experimental conditions are described under ``Experimental Procedures.'' B, dependence of the first-order rate constants (R^2 = 0.876) of the NO binding to the Fe wild-type (5 µM) on the NO concentration.



The k value (3.2 times 10^4M s) of NO to the E318D mutant was about 2-fold higher than the wild-type (1.7 times 10^4M s) (Table 2). 7-Ethoxycoumarin, anthracene, and phenanthrene markedly increased the k value of the E318D mutant without substrate up to 6.2-fold. Dibenzanthracene decreased the k value of the mutant by 4.5-fold (Table 2).

The k value of NO for the T319A mutant was 3.8 times 10^4M s. This is similar to that of the E318D mutant.

Calculated k values of NO from the wild-type P450 1A2 in the presence of phenanthrene or dibenzanthracene were much larger than in the absence of the substrates. Calculated k value from the E318D mutant was 3.6-fold higher than the wild-type. Addition of 7-ethoxycoumarin, anthracene, and phenanthrene increased the k value by 5.2 8.9-fold, whereas dibenzanthracene did not change the k value (Table 2).

Quantum yield for the laser-induced photodissociation of NO from P450 1A2 enzymes was about 0.01 under present conditions. We only compared relative quantum yield under specific conditions (Table 4). Relative quantum yields of the wild-type and the E318D mutant enzymes with dibenzanthracene were higher than those observed for other enzyme solutions by 7.2- and 3.5-fold, respectively (Table 4).




DISCUSSION

The K(d) value of the axial ligands to the hemoprotein will reflect the structure of the final bound state(30, 31) . 7-Ethoxycoumarin and anthracene markedly decreased the K(d) value by 3.4-fold and by more than 37-fold, respectively. Phenanthrene and dibenzanthracene markedly increased the K(d) value by more than 8-fold (Table 2). Perhaps the Fe-N-O bond angle in P450 1A2 may not be 180°, and is slightly bent due to distal amino acid constraint, even without any substrates. It seems that 7-ethoxycoumarin and anthracene bindings at or near the distal site of heme may partially release this constraint, leading to a more linear Fe-N-O bond. In contrast, bindings of other larger hydrocarbons such as phenanthrene and dibenzanthracene, that have an extra benzene ring(s) on the side of the long axis, may further distort the less linearity of the Fe-N-O bond. L-Arg and N^G-hydroxy-L-Arg are rather flexible and have an ionic character in contrast to other hydrocarbon substrates studied here. Thus, effects of these flexible and ionic substrates on the K(d) value will be less pronounced than other aromatic hydrocarbon substrates (Table 2).

Distal site structure of P450 1A2 is altered by the E318D mutation, and thus effects of those substrates on the K(d) value were less marked in the mutant than the wild-type. Nevertheless, dibenzanthracene most increased the K(d) value among substrates studied here (Table 2).

The NO-E318A mutant complex was easily reduced. NO-metHb complex and NO-hemin complex in polar solvents are also easily reduced(23, 24) . NO has a strong electron donating character known to be a 3-electron donor (46) . In the NO-metHb complex, presumably an ionic bridge consisting of distal His, NO, and heme iron is formed (Fig. 5A)(23, 24, 25, 26, 27, 28, 29) . Also, a polar solvent will form an ionic bridge with NO and heme iron. This special ionic bridge consisting of the Fe-N-O-H bond may facilitate the iron reduction in these NO-Fe complexes by forming a proper Fe-N-O orientation to push electrons. On the other hand, the carboxyl group of Glu-318 may make a different ionic bridge consisting of Fe-N-O-H at P450 1A2 in the way of forming an improper Fe-N-O orientation to reduce the heme iron (Fig. 5B). This postulate bridge is possible, although it does require postulation of an effectively low pK(a) of Glu-318. The cysteine thiolate of the P450 1A2 proximal site should push electrons to the heme iron and facilitate the iron reduction. In the NO-wild-type P450 1A2 complex, however, the iron reduction may be in part canceled out by electrons from both proximal and distal (even slightly) sites. In the NO-E318A mutant complex, there is no more ionic bridge in the distal site and thus a strong electron donating character of cysteine thiolate or NO may be manifested. It is also possible that the distal site conformational change caused by the mutation alters the polarity of the heme environment and/or redox potential of the heme iron.


Figure 5: Hypothetical heme active sites of Hb (A) and P450 1A2 (B) in the presence of NO.



NO-heme (Fe) reduction of Hb was explained by a mechanism (47) as: Fe + NO (Fe NO); (Fe NO) + 2OH (Fe NO(2)) + H(2)O; (Fe NO(2)) + NO (Fe NO) + NO(2). H(2)O or alcohol is possible to participate in the Fe-NO reduction with such a mechanism (48) as shown in the following (B, base),

The E318A mutant is the low-spin state and is likely that H(2)O or OH is located near the distal site (30, 49) . Since the redox change of the NO-P4501A2 complex appears to be associated with the spin state of the mutant, accessibility of hydroxide ion to NO may also contribute to the redox stability of the NO-Fe complex.

Larger hydrocarbons such as phenanthrene and dibenzanthracene may largely distort the Fe-N-O conformation in the wild-type in terms of the K(d) value (Table 2). Similar marked distortion caused by these hydrocarbons must also take place at the distal site in the E318A mutant and disturb the Fe-N-O reduction in the mutant (Table 3).

The NO-ferrous forms of the wild-type and E318A mutant enzymes have a Soret absorption at 400 nm (Fig. 2A and Fig. 3), suggesting that 5-coordinated complexes with only NO as an axial ligand are formed (20, 21, 22, 50, 51) . NO is tightly bound to Fe-heme, and thus the Fe-NO bond has a covalent character(46) . As a result, the trans thiolate bond with Fe will be dissociated to compensate the electron density at Fe.

The change of the k value will reflect the change of the access channel of NO in P450 1A2(31). All substrates studied here may release steric constraint at the NO access channel and/or enlarge the NO access channel. In accordance with this, relative quantum yield of the photoinduced dissociation of the NO-P450 1A2 complex was increased by the addition of the substrates (Table 4). Dibenzanthracene increased the quantum yield mostly among the substrates studied. Dibenzanthracene may distort the approximate linearity (but not an angle of exactly 180°) of the Fe-N-O bond and simultaneously the NO access channel by limiting the distal space for the ligand approach to the heme iron. Thus, the quantum yield of the NO complex in the presence of dibenzanthracene is the highest among those in the absence and presence of other substrates.

In conclusion, 1) conserved Glu/Asp in the distal site of P450 1A2 (and presumably of NOS) must be crucial in the redox stability of the NO-ferric complex; 2) non-linear aromatic hydrocarbons, such as phenanthrene and dibenzanthracene, markedly change the kinetic values associated with the NO binding; 3) phenanthrene and dibenzanthracene also markedly disturbed the reduction of the NO-E318A mutant complex. The ionic bridge in the NO-P450 1A2 complex hypothesized here will require further studies to clarify this finding by comparing with P450nor (52) and metHb distal structures.


FOOTNOTES

*
This work was in part supported by a Grant from Sumitomo Science Foundation and Grants-in-Aid 7680670 and 7558083 from the Ministry of Education, Science, Sports and Culture of Japan (to T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute for Chemical Reaction Science, Tohoku University, Sendai 980-77, Japan. Tel.: 81-22-217-5604; Fax: 81-22-217-5604 (office), 81-22-217-5664 (library), or 81-22-263-9849 (library); shimizu{at}icrs.tohoku.ac.jp.

(^1)
The abbreviations used are: P450, cytochrome P450; P450 1A2, cytochrome P450 1A2, rat liver microsomal cytochrome P450, CYP1A2, or cytochrome P450(d); NOS, nitric oxide synthase; Hb, hemoglobin; NOR1, (D,L)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide; 7-ethoxycoumarin, 7-ethoxy-2H-1-benzopyran-2-one; dibenzanthracene, 1,2:3,4-dibenzanthracene; K, dissociation constant; k, rate constant.


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