A Positively Charged Cluster Formed in the Heme-distal Pocket of Cytochrome P450nor Is Essential for Interaction with NADH*

Takashi Kudo, Naoki Takaya, Sam-Yong ParkDagger , Yoshitsugu ShiroDagger , and Hirofumi Shoun§

From the Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan and the Dagger  RIKEN Harima Institute/SPring-8, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan

Received for publication, August 10, 2000, and in revised form, November 11, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arg and Lys residues are concentrated on the distal side of cytochrome P450nor (P450nor) to form a positively charged cluster facing from the outside to the inside of the distal heme pocket. We constructed mutant proteins in which the Arg and Lys residues were replaced with Glu, Gln, or Ala. The results showed that this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br-) perturbed the heme environment along with the reduction step in P450nor-catalyzed reactions, which was similar to the effects caused by the mutations. We determined by x-ray crystallography that a Br-, termed an anion hole, occupies a key region neighboring heme, which is the terminus of the positively charged cluster and the terminus of the hydrogen bond network that acts as a proton delivery system. A comparison of the predicted mechanisms between the perturbations caused by Br- and the mutations suggested that Arg174 and Arg64 play a crucial role in binding NADH to the protein. These results indicated that the positively charged cluster is the unique structure of P450nor that responds to direct interaction with NADH.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytochrome P450nor (P450nor)1 is involved in fungal denitrification as the nitric-oxide reductase (Nor) (1) that catalyzes the following reaction (Scheme 1).
<UP>2NO</UP>+<UP>NAD</UP>(<UP>P</UP>)<UP>H</UP>+<UP>H<SUP>+</SUP> → N<SUB>2</SUB>O</UP>+<UP>NAD</UP>(<UP>P</UP>)<SUP><UP>+</UP></SUP>+<UP>H<SUB>2</SUB>O</UP>

<UP>S<SC>cheme</SC> 1</UP>
P450nor can complete this reaction without the aid of other protein components such as P450 reductase and thus receives electrons directly from NADH. Furthermore, the high catalytic turnover (more than 1000 s-1 at 10 °C) (1, 2) is another notable property that may arise from direct interaction with NADH. The overall reaction can be divided into three partial reactions (2).
<UP>Fe<SUP>3+</SUP></UP>+<UP>NO → Fe<SUP>3+</SUP>-NO</UP>

<UP>S<SC>cheme</SC> 2</UP>

<UP>Fe<SUP>3+</SUP>-NO</UP>+<UP>NAD</UP>(<UP>P</UP>)<UP>H → </UP>I+<UP>NAD</UP>(<UP>P</UP>)<SUP><UP>+</UP></SUP>

<UP>S<SC>cheme</SC> 3</UP>

I+<UP>NO</UP>+<UP>H<SUP>+</SUP> → Fe<SUP>3+</SUP></UP>+<UP>N<SUB>2</SUB>O</UP>+<UP>H<SUB>2</SUB>O</UP>

<UP>S<SC>cheme</SC> 4</UP>
First, the ferric resting form (Fe3+) of P450nor binds the substrate NO to form a ferric iron-nitric oxide complex (Fe3+-NO; Scheme 2). Thereafter, Fe3+-NO is reduced with NADH to form a specific intermediate (I) with a Soret absorption band at 444 nm (Scheme 3) and finally reacts with the second NO to form the product, N2O (Scheme 4).

We isolated P450norA and P450norB from Fusarium oxysporum (3) and P450nor1 and P450nor2 from Cylindrocarpon tonkinense (4, 5). These isoforms differ in terms of intracellular localization (mitochondria and cytosol). These P450nor isoforms also have different levels of specificity against electron donors. P450norA, P450norB, and P450nor1 prefer NADH as the electron donor, whereas only P450nor2 prefers NADPH to NADH, indicating that they can discriminate between NADH and NADPH and thus contain an intramolecular site for binding a reduced pyridine nucleotide. We showed from the characterization of chimeric P450norB and P450nor2 proteins that the N-terminal half is more important than the C-terminal half for determining specificity against reduced pyridine nucleotides (5).

In contrast to the usual monooxygenase P450s that receive electrons from the heme-proximal side, we surmised that P450nor does so from the distal side. This characteristic should be reflected in the structure, although the crystal structure determined with respect to P450nor of F. oxysporum was fundamentally identical to those of other P450s (6). The most unusual structure outstanding in P450nor is that a wide open space is formed in the heme-distal pocket. We assumed that this space acts as the access channel for NADH (or NADPH). The crystal structure also revealed that the heme-distal pocket is in a highly hydrophilic environment compared with those of other P450s, which contain many water molecules (6, 7). A specific hydrogen bond network that comprises water-74, Ser286, water-33, and Asp393 is formed when NO binds to the resting heme (Scheme 2). Water-74 is adjacent to the heme iron-bound NO, and thus the network connects the active site to the bulk solvent and would act as a proton delivery system when the Fe3+-NO complex is reduced with NADH (Scheme 3). This heme-distal pocket abnormally concentrates positively charged Lys and Arg residues, suggesting that the positively charged cluster attracts the negatively charged NADH molecule by ionic interaction. We constructed a series of P450nor mutants by site-directed mutagenesis to examine the roles of the positively charged cluster and showed that the unique structure is probably responsible for the characteristic function of P450nor, namely direct interaction with NADH.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Mutagenesis and Expression Plasmids-- Plasmid preparation, digestion by restriction and modifying enzymes, and electrophoresis proceeded according to standard methods (8). An expression vector for P450nor (pT7-nor) comprised pRSET C (Invitrogen) with the T7 promoter in which the N-terminal His tag was replaced with the full length of P450nor cDNA (9). Site-directed mutagenesis of the P450nor cDNA was achieved using polymerase chain reaction (10) with pfp450-20 (9) as a template. M13-47 and M13-RV (Takara, Japan) were primers specific for the vector pUC18. The primers used to construct each mutant protein were as follows (mutated sites are underlined): K62A, CTGAAAAGCTCTCCGCGGTCCGCACTCG; R62E, CTGAAAAGCTCTCCGAGGTCCGCACTCG; R64E, CTCCAAGGTCGAGACTCGCCAAGGC; R64Q, CTCCAAGGTCCAGACTCGCCAAGGC; K77E, GCGCCAGTGGAGAGCAAGCAGCC; K81E, GCAAGCAGCCGAGGCAAAGCC; R174E, GAACGCCATTGAGACAAATGGTAG; R174Q, GAACGCCATTCAGACAAATGGTAG; R182E, GCTCCACTGCCGAAGAGGCCTCTG; K291E, GCACTAGCTATCGAGCGTACTGCC; K292E, CTAGCTATCAAGGAAACTGCCAAGG; R392E, CGCCTCTGAACGAAGATGTCGGAATC.

The resulting polymerase chain reaction product was inserted into pGEM-T (Promega), and the mutation was confirmed by sequencing the inserted nucleotide fragment. All plasmids expressing mutant proteins were constructed by replacing the BssHII-PstI fragment containing P450nor cDNA in pT7-nor with the corresponding portion of mutated cDNAs. The introduced mutations were again confirmed by sequencing the full length of the exchanged cDNA.

Expression-- The pT7-nor plasmid and derivatives were introduced into Escherichia coli JM109 (DE3). The transformed cells were cultured overnight at 30 °C in LA broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 25 µg/ml ampicillin) supplemented with 0.5% glucose (preculture). Precultures (20 ml) were inoculated into 2 liters of LA broth in a 5-liter Erlenmeyer flask with baffles and incubated at 30 °C with rotation at 120 rpm for 6-7 h. Cells were induced by an overnight incubation with 1 mM isopropyl 1-thio-beta -D-galactoside (IPTG).

Purification of Mutated P450nor-- The transformed cells were harvested and suspended in Tris buffer (20 mM Tris-HCl (pH 8.0), 0.1 mM dithiothreitol, 0.1 mM EDTA, 10% (v/v) glycerol) and then sonicated (200 watts, 10 min). The suspension was then centrifuged at 10,000 × g at 4 °C for 30 min, and then the supernatant was dialyzed against Tris buffer and centrifuged at 10,000 × g for 30 min. The supernatant was applied to a DEAE-cellulose (DE52, Whatman) column (bed, 30 ml) equilibrated with Tris buffer, and eluted with 0-0.4 M KCl gradient. The P450nor fraction was condensed by dehydration with polyethylene glycol and dialyzed against Tris buffer. The dialysate was then applied to a Mono-Q HR 5/5 column (Amersham Pharmacia Biotech) equilibrated with the same buffer and eluted with a 0-0.4 M KCl gradient. The P450nor fraction was condensed, dialyzed, and stored at 4 °C for further analyses.

Analytical Methods-- P450nor concentration was determined as reported (3) using a Beckman DU-7500 spectrophotometer. P450nor interaction with anions was observed by spectrophotometric titration. P450nor (10 µM) in 50 mM TES buffer (pH 7.2) was mixed with an equal volume (100 µl) of a KBr, KCl, or potassium phosphate solution in the same buffer in a cuvette (200 µl volume), and the spectrum was recorded.

The process by which NO binds to the resting ferric form of P450nor was observed by flash photolysis as reported (2). The reaction of P450nor with NADH was analyzed by following the appearance of the specific intermediate (Soret peak at 444 nm) upon reduction of the Fe3+-NO complex with NADH at 10 °C using a Unisoku rapid scan analyzer as described (2), except 50 mM TES buffer (pH7.2) was used instead of phosphate buffer.

The Nor activity of P450nor was assayed as reported (1). P450nor (4 nM) was incubated anaerobically with NO (55 µM) in the presence of 0.5 mM NADH, and the amount of N2O formed was determined by gas chromatography.

X-ray Crystallography-- Br- derivatives of P450nor were searched by co-crystallization (6). Co-crystals were grown in 10 mM KBr, 100 mM MES buffer at pH 5.6 using polyethylene glycol (PEG 4000) as a precipitant. The initial drops were composed of 2 ml of protein (45 mg ml-1) and 2 ml of precipitant solution that were equilibrated with precipitant in a 1-ml reservoir. Crystals obtained after 1 week at 293 K were suspended on a loop in a thin liquid film of stabilizing solution and frozen at 100 K directly in a cold nitrogen gas stream in a Cryostream Cooler (Rigaku Cryosystems). Data were obtained at the synchrotron radiation source of the BL44B2 (RIKEN Beam Line 2) station in SPring-8, Harima, Japan (11). All data sets were detected on R-AXIS IV imaging plates. Measurements were performed at 100 K and a wavelength of 1.0 Å. Diffraction data were integrated and scaled with DENZO (12) and SCALEPACK (12). The derivative crystal (P212121, unit cell parameters: a = 54.32, b = 78.05, c = 86.6 Å) was isomorphous to the native crystal. Data collection statistics are summarized in Table I.

Difference Patterson synthesis at 2.0 Å resolution calculated using the CCP4 package indicated two major binding sites in the asymmetric unit. The map shows Br- self-vectors on the Harker sections with more than 4 root-mean-square deviations of the map. The position of Br- was refined as (0.175 0.060 0.240 and 0.804 0.076 0.228) by vector space refinement (13).

The atomic model was built using the program TURBO-FRODO (14), and improved with SigmaA-weighted |2Fo - Fc| and |Fo - Fc| maps iteratively with X-PLOR 3.851 (15).The structures of the native P450nor enzyme in the orthorhombic space group (Protein Data Bank identification number 1ROM), without the coordinates of the water molecules, were used as an initial model for refinement of the derivative structures. After a first round of rigid body, simulated annealing and B-factor refinements, a |2Fo - Fc| map was calculated. After manually rebuilding the water molecules, other rounds of refinement were completed, which included positional, individual B factors and bulk solvent correction of the protein model. Water molecules were placed at positions where spherical densities were above 1.5 s in the |2Fo - Fc| map and above 3.0 s in the |2Fo - Fc| map and where stereochemically reasonable hydrogen bonds were allowed. Structural evaluations of the final models of the derivative enzymes using PROCHECK (16) indicated that 90.8% of the residues were in the most favorable regions of the Ramachandran plot and that no residues were in disallowed regions. The refined model included residues in each chain and 116 water molecules, yielding an R-factor of 21.0% and an Rfree-factor of 27.8%. The root-mean-square deviations in bond lengths and angles were 0.007 Å and 1.2°, respectively. The refinement statistics are summarized in Table I. Coordinates of the derivative P450nor have been deposited at the Protein Data Bank (identification code 1GED).


                              
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Table I
Crystal parameters, data collection, and structure refinement
Values in the outer shell are for the highest shell with a resolution of 2.09-2.0 Å.



    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Positively Charged Cluster on the Distal Side of P450nor-- The crystal structure revealed many Lys and Arg residues around or inside the heme pocket on the distal side of P450nor (6), as shown in Fig. 1. Among these positively charged residues, Lys77, Lys81, Arg182, and Arg392 are located on the surface of the entrance of the distal-heme pocket, and Lys62, Arg64, Arg174, Lys291, and Arg292 are buried inside the pocket. We constructed a series of mutant proteins by site-directed mutagenesis in which these five Arg and four Lys residues were replaced, respectively, with a negative charge (Glu) or a neutral (Gln or Ala) amino acid residue. We then investigated their properties to examine our hypothesis that this unique structure (positive charge cluster) is responsible for the direct interaction of P450nor with NADH. The results are summarized in Table II. On the other hand, the negatively charged groups, Asp88 and Asp393, are also located inside the pocket. The presence of so many charged groups along with many water molecules (7) makes the environment of the distal pocket highly positive and hydrophilic unlike those of other P450s. Therefore, this environment is very likely the structure that attracts negatively charged, hydrophilic NAD(P)H.



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Fig. 1.   Stereo view of the heme-distal pocket and the positively charged cluster of P450nor. Positively and negatively charged amino acid residues are shown in blue and red, respectively. Heme (violet) and charged residues are represented by the stick model. Other residues are illustrated by the ribbon model. Amino acid residues are represented by one-letter abbreviations.


                              
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Table II
Kinetic parameters of the P450nor mutants
Averaged values of experiments repeated at least three times are presented. Experimental error was below 20% with the exception of the spin state ratio (less than 10%). ND, not detected; ---, not determined.

Effects of Mutations on Overall Nor Activity-- All of these mutations, except that at Lys81, modulated overall Nor activity to various extents (Table II). Mutations at the residues inside the distal pocket (Lys62, Arg64, Arg174, Lys291, and Arg292) decreased the activity more than those outside the pocket (Lys77, Lys81, Arg182, and Arg392), and mutation to a negative charge (K62E, R64E) decreased the activity more than mutation to a neutral amino acid at the same position (K62A, R64Q). R174E, R174Q, R64E, and R292E completely lost overall activity, and mutations at Lys62 also considerably decreased the activity. All of these mutant proteins except R292E seemed to be folded correctly in heterologous E. coli cells because they formed complexes with carbon monoxide (data not shown). By contrast, R292E was formed as an unfolded protein without heme. This finding is reasonable because Arg292 corresponds to the conserved Arg residue (for example Arg299 in P450cam) that is essential for binding heme to the protein moiety (17). Arg292 of P450nor also interacts with one of the propionate side chains via a water molecule.2 Large amounts of these mutant proteins were expressed and purified for further characterization.

Spectral Perturbation by Mutations-- Fig. 2A shows that the ferric resting heme of native P450nor is in a mixture of high and low spin states (1). All of the mutations inside the pocket caused a reverse type-1 spectral change in the bound heme of each resulting mutant protein, that is, an increase in the ratio of the low spin state (Fig. 2B, Table II), whereas those at Lys77, Lys81, and Arg182, which are located outside the pocket, did not. The extent of the spectral perturbation did not correlate with the remaining overall activity but seemed to reflect the distance between the mutation site and heme. The mutations at Lys62 and Lys291, which are located close to heme, induced the largest perturbation, whereas mutations at Arg174, which is located further away from heme, caused a complete loss of the activity but only a weak perturbation. However, mutation at the side chain of Arg392, which is located on the entrance of the pocket and oriented toward the outside, caused moderate inactivation and spectral perturbation. Its neighbor, Asp393, is involved in the proton delivery network (6, 7). Therefore, the mutation of Arg392 may have perturbed this network, which in turn resulted in spectral changes in heme and partial inactivation of the enzyme.



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Fig. 2.   Optical absorption spectra of native (A) and R174E mutant (B) P450nor in a ferric resting state in the presence (solid line) or absence (dashed line) of 1.0 M KBr. Purified protein was dissolved in 50 mM TES buffer (pH 7.2).

Effects of Mutations on the Binding of NO-- Each mutant protein was further characterized by analyzing its partial reactions (Schemes 2-S4). During the first step of the Fe3+-NO complex formation (Scheme 2), all mutant proteins except R292E bound NO (data not shown). Their Fe3+-NO form afforded essentially the same spectra as native P450nor with a Soret peak at 431 nm (cf. Figs. 3 and 4). This binding process was observed by flash photolysis (2). This step is very rapid, and thus the minor modulation of the apparent rate constant (kobs) observed with each mutant (R64E, R64Q, and R174E; Table II) cannot be used to explain such a large decrease in the overall activity.



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Fig. 3.   Absorption spectral change during reduction (I formation, Scheme 3) observed with native P450nor by the rapid scan (A) or the stopped-flow (B) method. P450nor in Fe3+-NO (final, 5 µM) was mixed with NADH (final, 20 µM) anaerobically at 10 °C in 50 mM TES buffer. The gate time was set to 1 ms. The single exponential kinetic trace in panel B analyzed using the Guggenheim curve fitting estimated the value of kobs.

Effects of Mutations on the Interaction with NADH-- Rapid scan analysis (2) detected a spectral intermediate l, with a Soret peak at 444 nm, when the Fe3+-NO complex was mixed anaerobically with NADH, which is assumed to have arisen from the 2-electron reduction of Fe3+-NO with NADH (Scheme 3) (2, 18). Fig. 3 shows a spectral change typical of this process with native P450nor. Although the reduction step (Scheme 3) would be rate-limiting in the overall reaction (2), I can accumulate when the amount of NO added is similar to that of the enzyme (P450nor). Under such conditions I cannot react further with the second NO because no free NO remains, and thus I can be in a quasi-stable state suitable for accumulation (Fig. 3A) (2). Fig. 3B shows the value of kobs for the reduction (I formation) obtained from the time-dependent decrease in the absorbance at 427 nm, which is the isosbestic point of the spectrum of I and the resting enzyme Fe3+ (with 413 nm peak) (2). We found that kobs (reduction) for each mutant protein was decreased in parallel with the decrease in the overall activity (Table II). A typical spectral change during the reduction of a mutant protein (R64E) is shown in Fig. 4. When Fe3+-NO was reduced with a low concentration of NADH (Fig. 4A), I was undetectable. However, when the concentration of NADH was increased, a small amount of l was detected (Fig. 4B). These results indicate that since the formation rate of I of the mutant is very slow, it is comparable with the decomposition rate, resulting in no accumulation of I and only Fe3+ species formation (Fig. 4A).



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Fig. 4.   Spectral change during reduction of R64E mutant. Experiments were performed in the same manner as described in the legend to Fig. 3. NADH concentrations: A, 20 µM; B, 0.5 mM.

The results with other mutants were similar, showing that the decline in the overall Nor activity in each mutant arose from interruption of the reduction step because of the mutations. R174E and R174Q did not form I at all even after reduction with higher concentrations of NADH, which is consistent with their complete loss of Nor activity and highlights the crucial role of Arg174 in the interaction with NADH.

Effects of Mutations on the Interaction of I with the Second NO-- This process (Scheme 4) cannot be observed as an isolated reaction by rapid reaction analyses, unlike the former two steps. However, this step apparently was not modulated so much by each mutation. This was suggested from the finding that the amount of accumulated I as a result of the reduction of each mutant decreased in parallel with the decrease in kobs (reduction) (only the result obtained with R64E is shown in Fig. 4), which showed that the next step (Scheme 4) was much faster than the lowered reduction step. This conclusion is also consistent with the finding that the overall activity of each mutant decreased approximately in parallel with kobs (reduction) (Table II).

Interactions with Anions-- Halogen anions (19) or phosphate (present results) cause a reverse type-1 spectral change in the bound heme (Fig. 2A) and inhibit the overall reaction of P450nor (1). The Kd values obtained by spectrophotometric titration in TES buffer for bromide (Br-), chloride (Cl-), and phosphate were 100, 100, and 30 mM, respectively (data not shown). These anions inhibited the reduction step (Scheme 3). Fig. 5 shows the results obtained with only Br-. The half-inhibitory concentrations for Br- and Cl- were 50 mM (Fig. 5) and 120 mM (data not shown), respectively, which essentially agreed with the respective Kd values. These results demonstrated that the reduction step of P450nor was inhibited by the binding of these anions, which caused a decline in the overall Nor activity. Thus, the introduction of a negative charge(s) into the heme pocket by either mutation or anion binding exerted similar effects, that is, a reverse type-1 spectral change and inhibition of the reduction step.



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Fig. 5.   Inhibition of the reduction step (Scheme 3) of native P450nor by bromide. The kobs value was determined as described for Fig. 3B in the presence of the indicated concentration of KBr.

The effects of Br- were also examined with the mutant proteins with respect to the spectral perturbation. The spectrum of each of K77E, K81E, and R182E did not alter by the mutations. These mutant proteins were perturbed spectrally upon mixing with Br- to almost the same extent as the native protein (data not shown). By contrast, K62A, K62E, and K291E, which were already perturbed greatly by the mutations, were not further perturbed at all by Br- (data not shown). The mutations at Arg64 and Arg174 caused moderate spectral changes. Mixing these mutant proteins with Br- caused further reverse type-1 spectral change, as shown in Fig. 2B with the results on R174E. These results demonstrated that the mutations and the binding of Br- perturbed the heme environment in the same manner.

Identification of Br- Binding Sites-- Fig. 6 shows the Br- binding sites identified by x-ray crystallography. The most ordered Br- (Br1), located under the F-helix, was surrounded by the side chains of Asn241 and Ser150 and the backbone carbonyl of Thr168. This site is situated rather far from heme (Fig. 6A), beyond the middle of the I-helix where the helix is interrupted (6). Another ordered Br- (Br2) was buried deep inside the pocket in a space between heme and the polypeptide backbone near Ala289. The polypeptide backbone is bent upward at this position to create a space for Br- (anion hole).



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Fig. 6.   Stereo view of the heme-distal pocket of P450nor focused on Br- binding sites. A, ribbon diagram of heme-distal pocket of P450nor focused on Br-binding sites (red spheres). alpha -Helices are shown as blue coils and loops as yellow tubes. The temperature factors of the bromide ions were 29 and 30 Å2, respectively. Heme is represented by the ball-and-stick mode. This figure was produced using MOLSCRIPT (25). B, stereo view of the final electron density map (|2Fo - Fc|) for Br-. The map was calculated from reflections in the resolution range of 30.0 to 2.0 Å and is contoured at 1.5 s, where s is the S.D. of the electron density map. Atomic positions are indicated by specific colors: yellow, carbon; sky blue, nitrogen; and red, oxygen. The position of Br2 is represented by a red sphere. This figure was produced using TURBO-FRODO (14).



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Halogen (Br-) ions exerted effects that were apparently similar to those caused by mutations at Arg or Lys residues inside the heme pocket, that is, a reverse type-1 spectral change and inhibition of the reduction step. However, the extent of spectral perturbation by mutations was not proportional to that of concomitant inactivation (Table II) but seemed to depend on the distance of the mutation site from heme. Thus, introducing a negative charge into the neighborhood of heme would cause a larger spectral perturbation. Based on this assumption, the spectral change along with the inhibition of the reduction caused by Br- arose mainly from the binding of Br- to the site near heme (Br2 in Fig. 6; anion hole). The preference of anions as the binding site for this hole to the positive charge cluster composed of Arg and Lys residues suggests that the anion hole can attract a negative charge or an electron-rich moiety. This anion hole occupies a key spot in the pocket, neighboring heme, the terminus of the positively charged cluster (Lys62), and the terminus of the proton delivery network (Ser286). These results suggest that this anion hole is the location of electron and proton transfer to the Fe3+-NO complex (Scheme 3). If so, electron transfer from NADH should be interrupted if Br- occupies this hole. On the other hand, the spectral perturbation suggests that the hydrogen bond network around heme, which contains many water molecules (7), was altered or disrupted upon the binding of Br- or mutations. Such a misalignment of the network induced in the Fe3+-NO complex would also interrupt the electron transfer that must accompany the concomitant transfer of proton(s) supported by the hydrogen-bond network (7).

The discussion above implies that Br- bound at Br2 interrupts the electron transfer from NADH to heme rather than the binding of NADH. Conversely, the findings that mutations at Arg174 completely disrupted the reducing step but only slightly perturbed the heme environment (Table II) along with the results that the R174E mutant was further perturbed by Br- (Fig. 2B) are highly indicative that the mutant proteins (R174E, R174Q) were inactivated because their NADH binding ability was lost. Whether the binding or the electron transfer was disrupted could be confirmed if the Km for NADH and the maximum reduction rate could be compared between native and mutant proteins. However, we could not determine these kinetic constants with native P450nor because the reduction rate was too high even at 10 °C and was therefore beyond the capability of the instrument to analyze the rapid reaction.

The Lys and Arg residues examined in this study were distributed from outside the entrance of the heme-distal pocket to the vicinities of heme (Fig. 1). The present results showed that these positively charged amino acid residues are critically important for interaction with NADH, although the extent of their contribution to the interaction would vary depending on the role of each residue. All positive charges located outside the entrance would introduce NADH to the entrance. Four of the positive charges inside the pocket (Lys62, Arg64, Arg174, and Lys291) have side chains that are oriented toward the space of the pocket. The B'-helix and the loop structure between the F- and G-helices (F-, G-loop) forms the entrance gate of the pocket, and Arg64 and Arg174 are located just beneath B'-helix and the F-, G-loop, respectively. Their side chains are opposite each other (Fig. 1). The side chain of Lys291 extends upward from the bottom of the pocket, and its top reaches the vicinity of the side chain of Arg64. Thus, these residues form a positively charged barrier just beneath the entrance gate, which should immobilize the NADH molecule by placing between these residues the negatively charged pyrophosphate moiety of NAD(P)H. In addition, Lys62 should also be important for the interaction with NADH. Mutations at this site resulted in both an enormous decline of kobs (reduction) and a large spectral perturbation. Therefore, to identify whether electron transfer or binding was disturbed is difficult. Lys62 is located in the vicinity of heme within the positive cluster, and its side chain is oriented toward heme (Fig. 1). This side chain might orient an NADH molecule toward the correct direction when NADH accesses heme.

The positively charged cluster should be the novel motif for interacting with NADH that is responsible for the unique catalytic property of P450nor, namely direct interaction with NAD(P)H. The motif is in sharp contrast to the Rossman fold structure (20, 21). Comparisons of several crystal structures of P450 species indicate that substrate recognition site 1 (SRS-1) (according to Gotoh) (22), which contains a B'-helix, is the most variable structure among P450 molecular species (23, 24). This finding also appears to hold true for P450nor, as shown in Fig. 7. SRS-1 of P450nor contains 6 positive amino acid residues including 4 (Lys62, Arg64, Lys77, and Lys81) that we investigated in the present study and 3 negative groups (Glu71, Asp88, and Asp90); this is in contrast to the corresponding region of P450cam, which contains only 1 positive and 5 negative groups. The other essential residues, Arg174 and Lys291, are located in SRS-2 and SRS-5, respectively. Furthermore, Ser286 and Asp393, which are involved in the proton delivery system (6, 7) are also involved, respectively, in SRS-5 and SRS-6. The present results indicate that P450nor has evolved to fit an interaction with NAD(P)H rather than with an organic substrate to be hydroxylated by changing mainly the amino acid residues in SRS regions. The results of the present study and others indicate that P450nor receives electrons from NADH on the distal side in contrast to other monooxygenase P450s that receive electrons on the proximal side.



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Fig. 7.   Alignment of the primary structures of SRS-1 containing the B'-helix of cytochromes P450cam and P450nor. Positively and negatively charged residues are shown in blue and red, respectively. The extent of the B'-helix is indicated only for P450nor.



    FOOTNOTES

* This study was supported by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN), Grant-in-aid 11116205 for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan, and the Sakabe Project of the High Energy Accelerator Research Organization (KEK), Tsukuba, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1ROM and 1GED) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ To whom correspondence should be addressed. Tel.: +81-298-53-4603; Fax: +81-298-53-4605; E-mail: p450nor@sakura.cc.tsukuba.ac.jp.

Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007244200

2 S.-Y. Park, H. Shoun, and Y. Shiro, unpublished data.


    ABBREVIATIONS

The abbreviations used are: P450nor, cytochrome P450nor; NO, nitric oxide; Nor, nitric-oxide reductase; TES, N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid; MES, 2-morpholinoethanesulfonic acid; Br-, bromide.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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