©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Spectroscopic and Kinetic Studies on Reaction of Cytochrome P450nor with Nitric Oxide
IMPLICATION FOR ITS NITRIC OXIDE REDUCTION MECHANISM (*)

(Received for publication, August 4, 1994; and in revised form, November 9, 1994)

Yoshitsugu Shiro (§) Motoyasu Fujii (¶) Tetsutaro Iizuka Shin-ichi Adachi Koki Tsukamoto Kazuhiko Nakahara Hirofumi Shoun

From the  (1)Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan (2)From the College of Science and Technology, Nihon University, Chiyoda-ku, Tokyo 101, Japan (3)From the Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H N(2)O + H(2)O + NAD). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(FeNO). Reduction of P450nor(FeNO) with NADH yielded an intermediate, which transiently formed ( = 100 ms) and spontaneously decomposed to the Fe state. The optical absorption spectrum of the intermediate was different from that of P450nor(FeNO), which was formed by either a one-electron reduction of P450nor(FeNO) with Na(2)S(2)O(4) or NO binding to P450nor(Fe). On the basis of these observations, we suggested that the intermediate is presumably a two-electron reduced product of P450nor(FeNO) by NADH, formally the (FeNO) complex. We determined the rate constants of these reactions at 10 °C for the NO binding to P450nor(Fe) (2.6 times 10^7M s), the NADH reduction of P450nor(FeNO) (0.9 times 10^6M s), and the spontaneous decomposition of the intermediate (0.027 s). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s at 10 °C), which we measured by monitoring the NADH consumption. Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover. On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.


INTRODUCTION

The cytochrome P450nor (^1)is a heme enzyme found in the denitrifying fungus Fusarium oxysporum(1, 2, 3) ; this fungus can reduce NO(3) and NO(2) to N(2)O. Shoun and his co-workers (4) isolated and purified this P450nor from F. oxysporum cultivated in the presence of NO(3) and NO(2). As identified by its name, P450, this enzyme contains protoheme in its active site as a prosthetic group, and its ferrous (Fe)CO complex exhibits a Soret absorption at 447 nm in its optical absorption spectrum. Its ESR spectrum in a ferric state also gives a typical feature of P450, in which a high spin signal at g = 8 and a set of low spin signals (g = 2.43, 2.25, and 1.91) are resolved at 4 K. (^2)All of these spectroscopic data are indicative of ligation of a thiolate anion from Cys as a fifth axial ligand of the heme iron. Furthermore, its cDNA analysis suggested that this enzyme can be involved in the superfamily of cytochrome P450s (5) (cytochrome P450 55A1 is its systematic name given on the basis of its cDNA sequence). Its amino acid sequence deduced from the cDNA analysis showed about 25% identity with that of Pseudomonas putida P450cam and about 40% with that of Streptomyces P450.

Despite these similarities in its structure and properties, the biological function of P450nor is very unique compared with other usual P450s. Although the usual P450 activates O(2) to catalyze a monooxygenation reaction, P450nor exhibits nitric oxide reductase (nor) activity. This enzyme can reduce NO to N(2)O but cannot catalyze the monooxygenation reaction. This means that in the denitrifying process by the fungus F. oxysporum, P450nor acts to rapidly diminish poisonous NO produced in the cell by reducing it to N(2)O. In this P450nor reaction, it was also noted that electrons required for the NO reduction are directly transferred from NADH to P450nor, in sharp contrast with the monooxygenase reaction by other usual P450s, where the electrons are donated from NAD(P)H to the enzyme through a flavoprotein or an iron-sulfur protein. No redox co-factor has been detectable in P450nor except for protoheme, in contrast with some flavo-hemoproteins such as P450(6) , NO synthase(7, 8) , and so on.

Shoun and his co-workers (4) have defined the stoichiometry and the turnover number of the reaction catalyzed by P450nor from the product analysis with gas chromatography as follows.

This reaction was hardly inhibited by CO, implying that a ligand-free Fe state is not involved in the P450nor catalytic cycle. It must also be noted that the ligand-free Fe form of P450nor cannot react with NADH, but its ferric NO-bound form can. On the basis of these observations, Shoun et al.(4) proposed a molecular mechanism for the NO reduction by P450nor as is shown in Fig. SI. In this hypothetical mechanism, NO binds to the Fe state of the enzyme to yield the FeNO complex. It is then reduced with ``one electron'' from NADH to be converted to the FeNO form. In this stage, the electron moves from the iron to the ligand NO, and subsequently the resultant FeNO complex spontaneously decomposes to the Fe state, releasing NO. The NO produced is non-enzymatically coupled with another NO to form N(2)O(9) .


Scheme I:


In this hypothetical mechanism, however, we have two problems. First, NADH acts as a one-electron reductant, although it generally donates two electrons to an acceptor in the form of a hydride anion. Second, we have never identified the FeNO complex of P450nor because it is too unstable at room temperature to be detected spectrophotometrically. In the present study, we examined the following three reactions using stopped-flow rapid scan, flash-photolysis, and low temperature spectroscopic methods.

On the basis of the spectroscopic and kinetic data, we propose a possible mechanism for the NO reduction to N(2)O catalyzed by P450nor.


MATERIALS AND METHODS

Sample Preparation

Cytochrome P450nor was isolated from F. oxysporum and purified by the method described elsewhere(4) . The concentration of the enzyme for the kinetic and the spectroscopic measurements was about 5 µM, which was spectrophotometrically determined using = 100 mM cm for the FeNO enzyme. The buffer used for the kinetic measurements was 0.1 M sodium phosphate at pH 7.2, and that for the low temperature spectral measurements contained 40% ethylene glycol as an antifreeze. To prevent NO(2) formation, the buffer solution for the spectral and the kinetic measurements was thoroughly purged of air by N(2) gas before adding NO. NO gas was introduced into the sample solution after passing 1 M KOH and the buffer solutions. The reduced form of NADH was purchased from Sigma and was used without further purification. It was dissolved in 50 mM borate buffer, pH 9.2, and its concentration was spectrophotometrically determined using = 6.22 mM cm at 340 nm.

Determination of Turnover Number in P450nor Catalytic Cycle

The turnover number in the NO reduction reaction catalyzed by P450nor was determined by monitoring the NADH consumption rate under various concentrations of NO, where the NO concentration was controlled by bubbling the NO/N(2) mixed gas produced with the gas divider (ESTEC SGD-SC-0.5L). The buffer solution containing NO (2.5, 2.0, 1.6, 1.5, 1.2, or 1.0 mM) and NADH (0.16 mM; 1 of absorbance at 340 nm) in the optical cell was incubated at 10 °C for 5 min. The reaction was started (t = 0) upon injection of P450nor into the optical cell; the final concentration of the enzyme was 5.4 nM. The rate of the NADH consumption in the catalytic cycle was monitored by the absorption change at 340 nm. The reciprocal of the initial rate in the NADH consumption per mole of the enzyme (v/e) at each NO concentration was plotted against the reciprocal of the NO concentration (Lineweaver-Burk plot). The turnover number and the Michaelis constant (K(m)) in this reaction were calculated from the y intercept and the x intercept in this plot, respectively.

Low Temperature Absorption Spectral Measurements

Visible absorption spectra were measured using a Hitachi U-3000 spectrophotometer, equipped with a handmade temperature control unit. The temperature at the cell was electronically controlled with thermomodule elements (Netsu Denshi Co. Ltd.) and was monitored by a platinum electrode.

Flash Photolysis Measurement

The NO binding to the Fe form of P450nor was examined by a flash photolysis experiment, which was performed using the second harmonic (532 nm) of a Q-switched Nd:YAG laser (Surelite I, Continuum), producing an excitation flash of 220 mJ with a pulse width of 4.6 ns. The beam spot was transformed into an ellipsoid using a cylindrical lens and was allowed to fall on the quartz window (2 times 10 mm) of the observation cell. The absorption spectra after photodissociating NO from the heme iron were measured by a multichannel photodiode array. The light pulse for recording the spectrum was generated by a pulse generator (PG-230, Hamamatsu Photonics) and a high speed gated image intensifier unit (C2925-01, Hamamatsu Photonics) after sensing a portion of the laser pulse for photolysis. The time course of the absorption change at a single wavelength was monitored by a monochromator with a photomultiplier. Traces from a number of measurements were accumulated on the oscilloscope (DS-8631, Iwatsu), and the average trace was transferred to a personal computer (PC98DX2, NEC) via a General Purpose Interface Bus interface. All equipment was constructed by Unisoku (Osaka, Japan). The association rate constant of NO to the ferric enzyme was measured under the pseudo-first order reaction condition ([P450nor] [NO] = 2.5 mM at 10 °C), assuming that the dissociation rate constant is small enough, compared with the association rate constant.

Stopped-Flow Measurement

The stopped-flow rapid scan measurements were carried out using the apparatus constructed by Unisoku. Before the measurement, the buffer in the reservoir was thoroughly treated with bubbling N(2) gas, and the degassed buffer was passed many times through the mixer and the optical cell. In the measurement with our apparatus, the dead time was 6 ms, which is the time for allowing the sample solution to flow from the mixer to the optical cell. For the measurement of the single turnover reaction, the buffer containing dissolved NO was added to the Fe enzyme solution in the reservoir, where its final concentration was about 5 µM. The temperature of the sample was controlled by a circulating thermostated water around the reservoirs, the mixer, and the optical cell with RM6 (Lauda).


RESULTS

Turnover in P450nor Reaction

We first determined the turnover number in the NO reduction reaction catalyzed by P450nor at 10 °C by spectrophotometrically monitoring the NADH consumption rate under various NO concentrations. As shown in Fig. 1, the absorption of the reduced form of NADH at 340 nm was dramatically decreased in the presence of NO upon addition of P450nor, showing the consumption of NADH in the P450nor catalytic reaction. We obtained the initial rate of the NADH consumption from the slope at t = 0 at each NO concentration (1.0 2.5 mM) and plotted it in the form of a double reciprocal (Lineweaver-Burk) plot, as shown in the inset in Fig. 1. From this plot, we estimated the turnover number at 1200 s, and K(m) for NO at 0.6 mM in the NO reduction at 10 °C.


Figure 1: Time course of the absorption change of the reduced form of NADH at 340 nm in the NO reduction reaction catalyzed by P450nor. The optical cell containing 2.5 ml of the NADH solution (about 0.16 mM (A = 1)) was treated with bubbling N(2)/NO mixed gas for 15 min. After incubation at 10 °C for 5 min, recording of the absorption change was then started. Upon adding 30 µl of P450nor solution (0.45 µM), the reaction was started (t = 0), as shown by the arrow in the figure. In the catalytic reaction, the reduced form of NADH was consumed, resulting in a decrease in the absorption at 340 nm. The initial rate (v) was obtained from the slope at t = 0. Inset shows the double-reciprocal plot (e/v versus 1/[NO]). From the y and the x intercepts in this plot, the turnover and K values were estimated, respectively.



The turnover (1200 s) at 10 °C we obtained here is seemingly larger than that (500 s) at 30 °C previously reported by Nakahara et al.(4) , which was determined by the product analysis with gas chromatography. However, because such a fast reaction is over the limitation of measurement with their method(4, 10) , they suggested that the real value might be much larger than 500 s at 30 °C.

NO Binding to Ferrous (Fe) State

We measured the optical absorption spectra of P450nor in various oxidation/ligation states using the rapid scan stopped-flow technique. The spectra of the stable compounds in the Fe, Fe, and FeNO states of the enzyme were obtained by mixing each enzyme solution with degassed buffer. The spectra obtained (Fig. 2A) were the same as the corresponding ones measured statically by a conventional spectrophotometer, which had been reported elsewhere(4) , showing the reliability of this method for the spectral measurement.



Figure 2: Absorption spectra of P450nor in various oxidation/ligation states measured at 10 °C with the stopped-flow rapid scan method. A, a ligand-free Fe, a NO-bound Fe, and a ligand-free Fe; B, a NO-bound Fe, a NO-bound Fe, and its NADH-reduced (I) states. The spectra of the ligand-free Fe, the FeNO, and the ligand-free Fe enzymes are the same as the corresponding ones measured by conventional spectroscopy. The spectrum of the FeNO complex was obtained about 50 ms after mixing of the Fe enzyme with the NO-dissolving buffer. It is indistinguishable from that obtained upon mixing the FeNO complex with Na(2)S(2)O(4) solution. P450nor(FeNO) was stable for about 100 ms after the formation but was gradually converted to the Fe state. Formation of the NADH-reduced product of P450nor(FeNO) (I) is described in the legend of Fig. 4and Fig. 6.





Figure 4: A, absorption spectral change of P450nor(FeNO) 836 ms after mixing the enzyme solution with NADH (100 µM) in the stopped-flow experiment at 10 °C. The spectra measured at 8, 13, 16, 21, 26, and 36 ms after the NADH mixing are shown. The Soret band for the FeNO complex at 431 nm decreases in intensity, while that for the intermediate, I, at 444 nm concomitantly increases in intensity. An isosbestic point is located at 440 nm. The gate time for the measurement was 1 ms. B, its difference spectral change based on the spectrum at 8 ms after the mixing. C, absorption spectral change 0.4204.9 s after the NADH mixing. A new isosbestic point is observed at 427 nm. The Soret absorption for I at 444 nm decreases in intensity, while that for the Fe enzyme at 413 nm concomitantly increases in intensity. D, its difference spectral change based on the spectrum at 0.4 s. In these measurements, the NO concentration was set to be a small excess amount of the enzyme concentration (5 µM).




Figure 6: A, time course of the absorption change at 427 nm after mixing P450nor(FeNO) with NADH (from the FeNO to I) measured at 10 °C by the stopped-flow apparatus. In this measurement, the NO concentration was set to be almost equal to the enzyme concentration (5 µM). Under this condition, we could obtain the single exponential kinetic trace shown here. We estimated the apparent rate constant (k) at ambient NADH concentration by analyzing this kinetic trace with the Gugenheim curve-fitting method. Inset shows a plot of the k value against the NADH concentration employed. By analyzing the NADH concentration dependence of k, we estimated the bimolecular reaction rate constant (k(2)) at 0.9 times 10^6M s. B, time course of the absorption change at 440 nm from I to the Fe state at 10 °C. The kinetic trace is a single exponential. The apparent rate constant, k, in this reaction is independent of the NADH concentration, as shown in the inset, and we then estimated the unimolecular reaction rate constant (k(3)) at 0.027s. C, time course of the absorption change at 427 nm in the presence of a large amount of NO at 10 °C. Tracea was recorded at 1 mM of the NO concentration. As NO was purged with bubbling N(2), tracesb-e were recorded. Finally, when the NO concentration became to be almost equal to the enzyme concentration (3 µM), the trace in the single exponential feature was recorded (see A).



Next, we tried to obtain the spectrum of the NO-bound ferrous form (P450nor(FeNO)) by the same method. Due to its instability at room temperature, the spectrum of P450nor(FeNO) has never been observable by the conventional spectrophotometric method. Using the stopped-flow technique, we succeeded in measuring the spectrum of P450nor(FeNO), which was formed by binding NO to the Na(2)S(2)O(4) reduced enzyme (P450nor(Fe)) or by reduction of P450nor(FeNO) with Na(2)S(2)O(4). In the spectrum of P450nor(FeNO) (Fig. 2B), we observed the diminished Soret absorption at 434 nm and the single broad visible band at 558 nm. The same spectrum was obtained with the low temperature (-10 °C) spectral measurement (data not shown). The spectral features resemble those of the ferrous NO complex of P. putida P450cam (P450cam(FeNO)), in which the Soret and the visible absorption are at 438 and 557 nm, respectively(11, 12) .

The NO binding to P450nor(Fe) was so fast even at low NO concentration (5 µM) and at 10 °C that we could not follow it by the stopped-flow measurements. The rate constant (k(0) in ) was estimated to be larger than 10^7M s.

NO Binding to Ferric (Fe) State

To obtain the NO binding rate constant to P450nor(Fe) (k(1) in ) with the flash photolysis method, we tested the photodissociation of the bound NO from the ferric iron (Fe). As is illustrated in Fig. 3A, the difference spectrum between the states before and immediately after the laser irradiation to P450nor(FeNO) was the same as that between P450nor(Fe) and P450nor(FeNO), where the peak, the trough, and the isosbestic point were observed at 391, 434, and 427 nm, respectively, indicating that the bound NO in P450nor(FeNO) was photodissociable from the Fe to yield P450nor(Fe). On the NO rebinding, the spectrum of the NO-photolyzed enzyme was restored to that of P450nor(FeNO).


Figure 3: A, spectral change of P450nor(FeNO) after the laser irradiation in the flash photolysis experiment. The spectra measured at 1, 5, 10, 15, and 30 µs after the flash was shown. The gate time was set to be 1.5 µs for the measurement. B, time course of the absorption change at 434 nm in the flash photolysis experiment. In this measurement, the NO concentration was 2.5 mM at 10 °C.



Using the time course of the absorption change at 434 nm (Fig. 3B), which was a single exponential decay, we estimated the rate constant for the NO binding to the Fe state (k(1)) to be 2.6 times 10^7M s at 10 °C.

Reduction of Fe-NO State with NADH

We followed the reduction of P450nor(FeNO) with NADH () by the stopped-flow rapid scan method (Fig. 4), because this reaction is a key process in the NO reduction by P450nor. During 30 ms after mixing the enzyme and the NADH solutions, the spectrum of P450nor(FeNO) changed into a new one having a Soret peak at 444 nm and a broad visible band at 544 nm with an isosbestic point at 440 nm (Fig. 4, A and B). After that, we observed another spectral change (Fig. 4, C and D) where the absorption at 444 nm decreased in intensity, with the concomitant appearance and increase in the absorption of the P450nor(Fe) at 413 nm with an isosbestic point at 427 nm. These spectral changes indicate that the 444-nm species is an intermediate (I) in going from the FeNO to the Fe states.

The intermediate, I, was also detectable with a low temperature spectroscopic method in the reaction of P450nor(FeNO) with NADH, as shown in Fig. 5. Although an absorption for the Fe enzyme is concomitantly observed at 413 nm, the spectral features are basically the same as those measured by the stopped-flow rapid scan method. When we compared the spectra of I and P450nor(FeNO) in Fig. 2B, we noted that the Soret (445 nm) and visible (544 nm) absorption positions of I are substantially different from those of P450nor(FeNO) (434 and 558 nm); I is formed on reduction of P450(FeNO) with NADH, while P450nor(FeNO) is formed on reduction with Na(2)S(2)O(4).


Figure 5: Absorption spectra of P450nor(FeNO) and its NADH-reduced product, I, at -10 °C measured by Hitachi U3000. In the spectrum of I, the absorption for the Fe form concomitantly appeared at 413 nm. In this measurement, the NADH and NO concentrations were about 100 and 40 µM, respectively. The spectral features were exactly the same as those measured by the stopped-flow rapid scan method (see the spectrum at 0.4 s in Fig. 4C). Even at -10 °C, the spectrum of I gradually converted to that of the Fe form.



The spectral change observed in the stopped-flow rapid scan measurements indicated that at least three states, i.e. the FeNO, I, and the Fe states of P450nor, are involved in its catalytic reaction and the enzyme changes during the catalytic cycle as shown in Fig. SII. To gain further insight into the P450nor catalytic mechanism, we then tried to obtain the reaction rate constant of each step under a single turnover condition, where the NO concentration was limited to be almost equal to the enzyme concentration.


Scheme II:


Under this condition, after a single reaction cycle from the FeNO to the Fe states through I, the reaction is supposed to stop because no more NO is present in the system, and the NO-free Fe state of P450nor can never be reduced with NADH.

We spectrophotometrically monitored the FeNO I reaction at 427 nm, which is the isosbestic point between I and the Fe states, and obtained the single exponential decay of the absorption (Fig. 6A). Because the decay was dependent on the NADH concentration, as shown in inset of Fig. 6A, we estimated the second order rate constant (k(2) in ) at 0.9 times 10^6M s at 10 °C. On the other hand, the rate constant of the spontaneous decomposition of I to the Fe states (k(3) in ) was estimated from the time course of the absorption change at 440 nm, which is the isosbestic point between the FeNO state and I. As shown in Fig. 6B, the change is also a single exponential decay but independent of the NADH concentration, so that we obtained the unimolecular rate constant (k(3)) at 0.027 s at 10 °C.

When we measured the spectral change (Fig. 4) and the kinetics (Fig. 6, A and B) of P450nor(FeNO) in the reaction with NADH, we set the NO concentration to be almost equal to the enzyme concentration (a stoichiometric reaction condition). It is because the effect of the very fast catalytic turnover on the spectral and the kinetic measurements must be excluded. Indeed, when we observed the NADH reduction of P450nor(FeNO) at 427 nm (Fig. 6C) in the presence of a large amount of NO (1 mM), the kinetic trace did not exhibit a normal single exponential feature and changed depending on the NO concentration. When the spectral change was followed under the same condition (data not shown), we observed the change of the spectrum of the FeNO state to that of the Fe states without detecting the spectrum of I, indicating that the kinetic trace in Fig. 6C corresponds to the direct conversion of P450nor(FeNO) to P450nor(Fe). These observations will be discussed in more detail in the ``Discussion'' section.


DISCUSSION

In the present study, we spectroscopically and kinetically examined the reactions of P450nor with NO and NADH and proposed a possible reaction mechanism for the NO reduction to N(2)O catalyzed by P450nor, as shown in Fig. SII. The catalytic cycle consists of three elementary steps: NO binding to the Fe enzyme (step 1), reduction of the FeNO enzyme with NADH (step 2), and conversion of the NADH-reduced product (I) to the Fe enzyme (step 3). In this proposed mechanism, we obtained two interesting findings with respect to the intermediate I, its absorption spectral property, and its spontaneous decomposition rate.

NADH-reduced P450nor (Reaction Intermediate I)

It was found that the absorption spectral feature of I, which is the NADH-reduced product of P450nor(FeNO), is significantly different from that of P450nor(FeNO) generated by the Na(2)S(2)O(4) reduction of P450nor(FeNO) (see Fig. 2B). This difference possibly reflects the difference in the NADH and Na(2)S(2)O(4) properties as an electron donor in the P450nor catalytic reaction. Indeed, we measured no obvious formation of N(2)O in the P450nor catalytic reaction using Na(2)S(2)O(4) instead of NADH, (^3)showing that Na(2)S(2)O(4) does not act as a real electron donor in the NO reduction reaction by P450nor. These observations imply that formation of I in step 2 is essential for the P450nor catalytic reaction.

In general, NADH donates two electrons to the acceptor, while Na(2)S(2)O(4) acts as a one-electron donor. On the basis of this biochemical knowledge, we could suggest that I is possibly the two-electron reduced product of P450nor(FeNO), formally the (FeNO) state. This electronic structure seems consistent with the absorption spectral feature of I; its Soret band (444 nm) is located at a longer wavelength than those of the FeNO state (434 nm) and of the FeNO state (431 nm), likely showing that a more negative charge resides on the FeNO moiety in I than in the FeNO and the FeNO states.

We also noted that the spontaneous decomposition of I to the Fe state is surprisingly slow. In the kinetic results, it was found that steps 1 (k(1) = 2.6 times 10^7M s) and 2 (k(2) = 0.9 times 10^6M s) are fast enough, in good consistency with the fast catalytic reaction by P450nor. However, the k(3) value (0.027 s) is too small to account for the large turnover number (1200 s). Therefore, some other factor must affect conversion of I to the Fe state, eventually resulting in the fast turnover.

As stated above, we measured the k(3) value under the condition of a stoichiometric reaction, where the NO concentration is almost equal to the enzyme concentration (about 5 µM). On the other hand, the catalytic turnover was measured in the presence of a large excess amount of NO (2.5 1.0 mM) relative to the enzyme concentration (5.4 nM). Because the k(3) value was independent of the NADH concentration (see Fig. 6B), the difference in the NO concentration is the only difference in the reaction condition affecting the k(3) and the turnover values. This fact suggests that excess NO significantly influences the decomposition rate of I to the Fe state.

Our above suggestion can be indirectly but reasonably supported by comparison of the kinetic and spectroscopic results in the reaction of P450nor(FeNO) with NADH under various amounts of NO. The kinetic traces in Fig. 6C are substantially different from those in the stoichiometric reaction (Fig. 6A), and their features depend on the NO concentration, qualitatively indicative of the effect of NO on the overall catalytic reaction. The kinetic feature in Fig. 6C could be explained as follows. The catalytic reaction turns over during the kinetic measurement, the FeNO state is constantly accumulated in the system, and amount of NO in the system governs the features of the overall reaction. Steps 1 and 3 are fast enough relative to step 2 during the catalytic turnover, so that the spectrum of I was undetectable under this reaction condition. Namely, the reaction in step 3 in the presence of large amounts of NO, i.e. the conversion of I to the Fe state in the catalytic cycle, is much faster than that in the spontaneous decomposition of I. It is likely to suggest that the decomposition of I ((FeNO) to the Fe state) is highly accelerated by another NO.

Mechanism Proposed for NO Reduction by P450nor

On the basis of the two novel considerations for I, the existence of the (FeNO) state and the acceleration of its decomposition, we propose a mechanism in which the two-electron reduced NO on the iron site reacts with another NO supported by two protons.

Although this reaction mechanism is speculative, the charge balance is chemically allowed in this reaction, and acceleration of the decomposition reaction of I by bulk NO can be qualitatively explained. In comparison, the usual P450 reaction can be represented as follows:

where the two-electron reduced O(2) on the iron site reacts with a substrate (RH), as we know. The P450nor and the usual P450 reactions are considered to be basically similar.

It is well known that the P450 monooxygenation reaction is supported by an acid catalyst located at the heme distal pocket, a hydroxyl group of the Thr residue, e.g. Thr-252 for P450cam(13, 14) . This Thr residue is highly conserved in all P450, and P450nor also has Thr at the 243 position in the heme pocket(5) . Therefore, Thr-243 is one possible candidate for the proton donor in the P450nor reaction. Furthermore, we found in the previous study that the channel to the heme pocket of P450nor is widely opened (15) for a second NO to have easier access to its heme pocket. All information on the structure and kinetic properties in the P450nor reaction appears to be consistently explained.

Other NO reducing heme enzymes have been found in other microorganisms such as Paracoccus denitrificans and Pseudomonas stutzeri(9, 10, 16) . For example, the NO reductase isolated from P. denitrificans by Hollocher and his co-workers (9) consists of one cytochrome b and two cytochrome c components (a cytochrome bc complex). For the NO reduction mechanism by this enzyme, they suggested a one-electron reduction of the FeNO moiety and NO release from the resultant ferrous complex, followed by dimerization of NO, protonation, dehydration, and N(2)O formation. The former process, i.e. the one-electron reduction of the FeNO moiety, is basically the same as the two-electron reduction of the FeNO complex proposed by us for the P450nor reaction. However, the NO release from the Fe state is not acceptable for our mechanism because the ligand-free Fe state is not involved in the P450nor reaction. Because the turnover by the NO reductase of P. denitrificans (27 s at 30 °C using phenazine methosulfate/ascorbate as an electron donor) is much slower than that of P450nor, acceleration of the reaction by bulk NO may not be necessary for the reaction by the P. denitrificans NO reductase.

In summary, we proposed the mechanism for the NO reduction to N(2)O catalyzed by P450nor, where the two-electron reduced complex of the FeNO reacts with another NO in the heme pocket to form N(2)O and H(2)O (see Fig. SII). This mechanism is novel and unprecedented, in comparison with the mechanism proposed so far for other copper- or iron-containing NO reductases. For example, in the NO reduction mechanism proposed for denitrifying enzymes by Averill and his co-workers(17, 18) , the iron- or copper-bound NO is converted to N(2)O via a metal-nitrosyl (M-NO) intermediate. However, this is not the case for P450nor because a nucleophile such as N(3) does not seriously affect the P450nor reaction(4) , indicating no involvement of the electrophile NO in the reaction.

The electronic structure of the intermediate I observed in the P450nor catalytic reaction is a clue to more clearly demonstrate the reaction mechanism. The (FeNO) state we proposed here has been rarely reported so far in hemoprotein chemistry, so we need to explore the reactivity, structure and electronic state of I in more detail. Because the life time of I is about 100 ms, it must be rapidly quenched after the reduction of P450nor(FeNO) with NADH for further spectroscopic studies such as ESR and resonance Raman measurements. These studies are now in progress in our laboratory.


FOOTNOTES

*
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan, by research funds from the Science and Technology Agency, Japan, and by the Biodesign Research Program from RIKEN (to Y. 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.

Present Address: Dept. of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan.

(^1)
The abbreviations used are: P450nor, P450 purified from F. oxysporum; P450, cytochrome P450; P450cam, P450 purified from P. putida; P450, P450 purified from Bacillus megaterium.

(^2)
Y. Shiro and M. Fujii, unpublished results.

(^3)
K. Nakahara and H. Shoun, unpublished results.


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