Phenol hydroxylase from Acinetobacter radioresistens S13 Isolation and characterization of the regulatory component Ersilia Griva 1 , Enrica Pessione 1 , Sara Divari 1 , Francesca Valetti 1 , Maria Cavaletto 2 , Gian Luigi Rossi 3 and Carlo Giunta 1 1 Dipartimento di Biologia Animale e dell’Uomo, Universita ` di Torino, Italy; 2 Dipartimento di Scienze e Tecnologie Avanzate, Universita ` del Piemonte Orientale, Alessandria, Italy; 3 Dipartimento di Biochimica e Biologia Molecolare, Universita ` di Parma, Italy This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron- sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cyto- chrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/ PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydro- phobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (abc)dimer.PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO. Keywords: regulatory proteins; multicomponent mono- oxygenase; phenol hydroxylase. Acinetobacter radioresistens S13 is able to grow on phenol as the sole carbon and energy source via the ortho-pathway (b-ketoadipate pathway). The first enzyme involved in phenol degradation is phenol hydroxylase (PH), a mono- oxygenase utilizing NADH as electron donor. In previous studies we have found that the enzyme is composed of three moieties which are readily separated by chromatographic steps: the oxygenase (PHO), composed of two heterotrimers (abc) (S. Divari, F. Valetti, P. Caposio, E. Pessione, M. Calvaletto, E. Griva, G. Gribaudo, G. Gilardi & C. Giunta, unpublished observation), the reductase (PHR) [1] and a third protein (PHI) that is described in this work. A similar molecular composition has been found in phenol hydroxylases from Pseudomonas CF 600 [2] and A. calcoaceticus NCIB 8250 [3] and in toluene-2-mono- oxygenase from Burkholderia cepacia [4], as well as in the soluble methane monooxygenases (MMOs) from Methylo- coccus capsulatus [5], Methylosinus trichosporium [6], Meth- ylocystis sp.M [7] and in alkene monooxygenase from Nocardia corallina [8]. In phenol hydroxylase of A. radioresistens S13, the third component is needed for the overall enzyme activity; in phenol hydroxylase from Pseudomonas CF 600, it promotes substrate–oxygenase interaction [9]; in MMOs it alters the local environment and the redox potential of the catalytic centre [6,10–12]. Interestingly, in other aromatic monooxygenases (i.e. toluene-4-monooxygenase from Pseudomonas mendocina [13], toluene/o-xylene monooxygenase from Pseudomonas stutzeri [14] and alkene monooxygenase from Xantobacter Py2 [15]), two proteins, rather than one, are present besides the oxygenase and the reductase moieties. In this case one has a regulatory function, the other is a Rieske-type ferredoxin. The question was asked whether, in A. radioresistens S13, PHI promotes the overall catalytic activity of the Correspondence to C. Giunta, Via Accademia Albertina, 13, 10123 Torino, Italy. Fax: + 39 0116704692, E-mail: carlo.giunta@unito.it Abbreviations: ANS, 8-anilinonaphtalene-1-sulfonic acid ammonium salt; CV, circular voltammetry; DPV, differential pulse voltammetry; MCD, magnetic circular dicroism; MMO, methane monooxygenase; MMOB, methane monooxygenase regulatory component; MMOH, methane monooxygenase hydroxylase; MMOR, methane mono- oxygenase reductase component; NBT, nitro blue tetrazolium; PH, phenol hydroxylase; PHI, phenol hydroxylase regulatory protein; PHR, phenol hydroxylase reductase; PHO, phenol hydroxylase oxygenase; T2M, toluene-2-monooxygenase. Enzymes: Phenol hydroxylase (EC 1.14.13.7); benzoate dioxygenase (EC 1.14.12.10); toluene 4-monooxygenase (EC 1.14.14.1); toluene 2-monooxygenase (EC 1.14.13 ); alkene monooxygenase (EC 1.14.13 ); xylene monooxygenase (EC 1.14.14.1); phthalate dioxygenase (EC 1.14.12.7); p-hydroxybenzoate hydroxylase (EC 1.14.13.2); toluene dioxygenase (EC 1.14.12.11); methane monooxygenase (EC 1.14.13.25). (Received 18 December 2002, accepted 6 February 2003) Eur. J. Biochem. 270, 1434–1440 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03505.x enzyme by: (a) PHI–phenol interaction, possibly facilita- ting substrate-binding to the active site of PHO; (b) PHI– PHR interaction, possibly resulting in an altered confor- mation of PHR more suitable for electron transfer to PHO; (c) PHI–PHO interaction, possibly causing a conformational change leading to the opening of the PHO active site. Materials and methods Bacterial strain The A. radioresistens S13 strain used in this work was isolated as previously described [16,17]. This bacterium bears several natural plasmids and is able to grow on either phenol or benzoate as the only carbon source. Culture conditions The culture media used were Luria-Bertani (LB) broth (peptone 10 gÆL )1 ,NaCl10 gÆL )1 , yeast extract 5 gÆL )1 )and the Sokol and Howell [18] minimal medium, where phenol was the only carbon source. The fed-batch fermentation procedure was used. The acclimation method was the same as previously reported [19]. Cells were harvested when growth reached the stationary phase and were stored frozen ()80 °C). Preparation of crude extract Cells were washed twice in 50 m M Hepes/NaOH buffer, pH 7.0, and then resuspended (1 g biomassÆmL )1 )in50 m M Hepes/NaOH buffer, pH 7.0. The biomass (about 200 g) was sonicated (Microsonix Sonicator Ultrasonic Liquid Processor XL2020) for a total time of 40 min at 20 kHz with intervals of 1 minute, keeping the cells on ice, and then centrifuged at 100 000 g for 1 h at 4 °C (ultracentrifuge LB60M, Beckman). The supernatant was assayed for phenol hydroxylase activity, that resulted to be present. This supernatant will be referred to as the enzyme crude extract. The pellet was further processed but no membrane-bound enzyme activity could be detected. Enzyme activity test Phenol hydroxylase activity was estimated polarographically modified from [2] by means of a Clark-type electrode (YSI Model 5300). The phenol hydroxylase reaction was moni- tored by evaluating the oxygen consumption due to PHO activity. The standard assay contained: 1.7 m M NADH, 100 lL of crude extract in 0.1 M Mops/NaOH buffer, pH 7.4 at 24 °C. The reaction was started by adding 1 m M phenol (Fluka). Both in the crude extract and after separation from the oxygenase, PHR activity was monitored by the reduction of cytochrome c in the presence of NADH at 550 nm [1]. Protein determination Protein content was determined by the Bradford test [20], using bovine serum albumin as standard. PHI purification An anion exchange DE-52 cellulose column (Whatman) (2.6 · 20 cm) was equilibrated with 50 m M Hepes/NaOH buffer, pH 7.0. The crude extract was eluted with a 0–0.5 M sodium sulfate gradient in 50 m M Hepes/NaOH buffer, pH 7.0 (final volume 1.1 L). This procedure allowed us to separate the oxygenase moiety. Fractions showing reduc- tase activity were applied on a second anion exchange column Source Q15 (Pharmacia) (1 · 5 cm) equilibrated with 50 m M Hepes/NaOH buffer, pH 7.0 containing 0.05 M sodium-sulfate. PHR and PHI were coeluted from this column with a 0.05–0.5 M sodium sulfate gradient in 50 m M Hepes/NaOH buffer, pH 7.0 (final volume 120 mL). After concentration by ultrafiltration (membrane Diaflo, cut off 3 kDa, Amicon), the enzyme-containing fractions (total volume 2 mL) were applied on a gel permeation Superdex 75-FPLC column (2.6 cm · 60 cm) (Pharmacia) equilibrated with 50 m M Hepes/NaOH buffer, pH 7.0, containing 0.05 M sodium sulfate to obtain separ- ation of PHR and PHI. All steps were performed at 4 °C. Monomers isolation by reverse-phase HPLC PHI was resuspended in 80 lL of 50% water/50% aceto- nitrile solution and 1% formic acid at a final concentration of 30 l M . The reaction was allowed to proceed at room temperature for 10 min modified from [21]. PHI monomers were purified using a HPLC Merk-Hitachi L6200 with a Diode Array L4500, equipped with a column Lichorosphere 100RP-8(Merk).Theflowratewas1mLÆmin )1 .The column was equilibrated with solvent A [water and 0.08% (v/v) trifluoroacetic acid] and the monomers were eluted using a linear gradient of 20–90% solvent B (water/ acetonitrile/trifluoroacetic acid 10 : 90 : 0.08, v/v/v) over 50 min. Hydrophobic interaction chromatography In order to inquire whether PHI could interact directly with phenol, PHI was dissolved in 50 m M Hepes/NaOH buffer, pH 7.0, containing 0.15 M sodium sulfate and was loaded on a Phenyl-Sepharose column (2.5 · 8cm)(Pharmacia) equilibrated in the same buffer. The flow rate was 2mLÆmin )1 . Molecular mass determination The molecular mass was determined by means of SDS/ PAGE, size exclusion chromatography and mass spectro- metry. SDS/PAGE was carried out in separating gels containing 15% acrylamide. The following proteins were used as standards: phosphorylase B (97 kDa), bovine serum albu- min (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21 kDa) and lysozyme (14 kDa). In addition, molecular mass peptide standards (Pharmacia) were used: globin (16.9 kDa), globin I + II (14.4 kDa), globin I + III (10.7 kDa) and globin I (8.2 kDa). Proteins were detected by silver staining. A Superdex 75-FPLC column (2.6 · 60 cm) (Pharma- cia) was equilibrated with 50 m M Hepes/NaOH buffer, Ó FEBS 2003 A. radioresistens S13 phenol hydroxylase regulatory component (Eur. J. Biochem. 270) 1435 pH 7.0, containing 0.05 M sodium sulfate. The column was calibrated with blue dextran 2000 and the following reference proteins (Pharmacia): bovine serum albumin (67 kDa), hen egg ovalbumin (43 kDa), chimotrypsinogen A (25 kDa) and bovine pancreas ribonuclease A (13.7), at 4 °C. The molecular masses of the calibration proteins were plotted semilogarithmically vs. the partition coeffi- cient K av to determine the apparent molecular mass of the sample. K av is defined as the ratio (V e ) V o )/(V t ) V o ). V e , V t , V o represent the elution, void and total column volume, respectively. The same experiment was repeated using 50 m M Hepes/NaOH buffer, pH 7.0, as eluent. PHI molecular mass was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral analysis, using a Biflex mass spectrometer (Bruker). The sample (3 nmol) was desalted, lyophilyzed and resuspended in 50 lL acetonitrile/water solution (70 : 30, v/v) and mixed with 50 lL sinapinic acid matrix. One lL of the resulting solution ( 30 pmol of PHI) was loaded. Isoelectric focusing The isoelectric point was determined by analytical IEF electrophoresis (Phast System, Pharmacia); the markers were those supplied by Pharmacia (pI calibration kit). NH 2 -terminal sequence After SDS/PAGE, the protein band was blotted onto Immobilon P (Millipore) membrane. The N-terminus was sequenced using the Applied Biosystems 470A automatic microsequencer, following the Edman degradation [22]. Optical spectroscopy The UV/Vis absorption spectrum of purified protein in 50 m M Hepes/NaOH buffer, pH 7.0, was determined from 200 to 700 nm using a DU-70 Spectrophotometer (Beck- man), at 20 °C. Fluorescence emission spectra of protein in the same buffer were collected at 20 °C, by means of a Luminescence Spectrometer LS 50 B-Perkin Elmer; using a 3-mL quartz cuvette (path length 10 mm). CD measurements were performed by a Jasco Spectro- polarimeter J-715 equipped with temperature-controlled Peltier Jasco PTC-348WI, using a 0.1-cm quartz cuvette. All spectra were recorded under nitrogen flow and the baseline was corrected by calibration with the dialysis buffer. The PHI concentration was 10 l M . Actual protein concentra- tions were verified by A 280 measurements made on CD samples. Spectra were recorded from 260 to 190 nm at a speed of 50 nmÆmin )1 , band-width of 1.0 nm, and a resolution of 0.1 nm in the temperature range between 10 °Cand70°C, after preincubation for 10 min at each temperature. Three runs were accumulated and averaged. CD measurements were reported as mean residue ellipticity, Q, in degreesÆcm 2 Ædmol )1 . Metal content The possible presence of an iron-sulfur cluster was inves- tigated by colorimetric analysis, following procedures modified from Lovenberg [23] and Beinert [24], respectively. Kinetic constants The catalytic activity of PHR was evaluated both in the presence and in the absence of PHI. K m and k cat were determined from Hanes–Haldane plot for the two electron acceptors cytochrome c and NBT, using 0.24 m M NADH as electron donor in 50 m M Tris/ sulfate buffer, pH 8.5, at 30 °C. Reconstitution of PH activity ‘ in vitro ’ Reconstitution of the complex from the purified fractions was studied by investigating the overall PH activity in the presence of variable amounts of each component. The assay was performed with a Clark type electrode in the presence of 1.7 m M NADH in 100 m M Mops/NaOH buffer, pH 7.4 at 24 °C. The basal oxygen consumption was subtracted from the consumption recorded after addition of 1 m M phenol. The effects of PHI and PHR concentrations on the overall PH activity were evaluated by systematic variation of PHI concentration (0.3; 0.6; 1.2 l M )overarangeof PHR/PHOratios(upto6),keepingfixedaPHOconcen- tration of 0.6 l M . Results PHI purification None of the fractions eluted from the first anion exchange column (DE 52-cellulose) exhibited the overall PH activity (i.e. oxygen consumption promoted by the presence of phenol). Individual fractions were tested for PHR activity, using cytochrome c as substrate. The fractions showing PHR activity were found to contain a second component that could be separated by gel permeation chromatography on Superdex 75, as shown in Fig. 1. The 18 kDa protein present in the elution pattern was identified as the PHI component on the basis of its ability to complement the Fig. 1. SDS/PAGE of PHI at different steps of purification. The numbers on the left represent the molecular masses (kDa). Lane A: low molecular mass standards; lane B: crude extract; lane C: after anion exchange chromatography on a DE 52-cellulose column; lane D: after anion exchange chromatography on a Source Q15 column; lane E: after gel filtration. The arrows point to PHR and PHI. 1436 E. Griva et al. (Eur. J. Biochem. 270) Ó FEBS 2003 PHO- and PHR-containing fractions in restoring the overall PH activity. The yield of the PHI component suggested that it accounts for 0.25–0.3% of the soluble cellular protein. Molecular mass and isoelectric point The molecular mass of PHI, determined by SDS/PAGE, was 10 kDa. A similar result (8.8 kDa) was obtained by mass spectrometry (MALDI-TOF) analysis (Fig. 2). A twice as large value (18 kDa) was found by gel-permeation chromatography on Superdex 75. Therefore, it is likely that the native protein occurs as a dimer. The isoelectric point, determined by analytical isoelectrofocusing on ampholyte gels, was 4.1. Absence of redox centres The UV/Vis absorption spectrum of PHI at pH 7.0 and at 20 °C exhibited the typical protein peak at 280 nm. Neither in native samples nor in samples treated with reducing or oxidizing agents were detected chromophoric groups absorbing in the interval between 300 and 800 nm. These results were confirmed by the Lovenberg [23] and Beinert analyses [24] which failed to show iron- or sulfur- containing redox-centres in the pure protein. In agreement with these findings, the emission spectrum of PHI, determined by spectrofluorimetry in the same conditions, exhibited a maximum at 345 nm on excitation at either 280 or 295 nm. N-terminal sequence The first 11 aminoacids at the N-terminus of PHI (sequence: SKVYLALQDND) were compared with the sequences of the so called ‘intermediate components’ from two other PHs. The N-terminal sequence of PHI from A. radioresis- tens S13 is identical (from residue number 3) to the sequence of the corresponding component of PH from A. calcoace- ticus NCIB 8250 (11/11 identity) [3] and very similar to that of the corresponding component of PH from Pseudomonas CF600] (8/11 identity) [25] (residue number 1 being the starting methionine). Secondary structure and thermal denaturation studies Figure 3 shows the far-UV CD spectrum of PHI in 10 m M sodium-phosphate buffer, pH 7.0. The CDNN deconvolu- tion programme indicates that this spectrum results from the presence of both helices and b-sheets. PHI was submitted to progressive heating. The CD spectrum was recorded in the temperature range 10–70 °C after reaching thermal equilibrium. As shown in the inset, the progressive decrease of the molecular ellipticity at k ¼ 200 nm (the absorption region of the peptide bond) reflects the occurrence of a transition between 35° and 55 °C. PHI does not interact with phenol The emission spectrum of the protein at 350 nm (on tryptophan excitation at 280 nm) is not affected by phenol addition. This result suggests that no interaction between phenol and PHI takes place. To confirm the lack of a hydrophobic site on the PHI surface, we investigated the possible interaction with the hydrophobic probe ANS: no fluorescence emission associated with ANS binding could be detected. Furthermore, PHI does not bind to a Phenyl- Sepharose column, confirming a low affinity for hydro- phobic sites in general. PHI is essential for the catalytic activity of the reconstituted PH system A stoichiometry 2 PHR monomers: 1 PHI dimer: 1 PHO (abc) dimer was found to provide optimal phenol reaction rates. PH activity in function of PHR concentration follows a Michaelian behaviour at fixed concentrations of PHO and PHI (Fig. 4). When the latter components are present at 0.6 l M , in terms of dimeric units, a maximum turnover number of 70 min )1 was obtained upon increasing PHR concentration: the plateau is reached at 1.2 l M PHR (in terms of monomeric units) (Fig. 4, continuous line with triangles). Excess of PHI over PHO does not alter the overall enzyme activity (Fig. 4, broken line with asterisks), in contrast to what observed in MMO from Methylosinus trichosporium [26]. The emission intensity of the PHR flavin shows a 17% increase after addition of PHI to either the complex PHR- PHO or to PHR alone, in the stoichiometry ratio PHR : PHO : PHI 2 : 1 : 1 (Fig. 5). On the contrary, the emission spectrum of PHO-bound ANS is not affected by the addition of PHI, suggesting no specific PHI interaction with the substrate binding site of PHO. PHI is not required for PHR activity towards alternative electron acceptors The kinetic constants for the two artificial electron acceptors cytochrome c and NBT, using NADH as the electron donor, were determined from Hanes–Haldane plot in the presence of either PHR alone or the couple PHR + PHI, as reported in Table 1. The differences in K m and k cat are not significant, suggesting that the catalytic activity of PHR does not depend on the presence of the regulatory protein. Fig. 2. MALDI-TOF spectrum of PHI. The protein was dissolved in 70% acetonitrile/water solution. Thirty pmol were mixed with 50 lL sinapinic acid matrix and were injected into the mass spectrometer. Ó FEBS 2003 A. radioresistens S13 phenol hydroxylase regulatory component (Eur. J. Biochem. 270) 1437 Discussion The small size of the PHI monomer ( 9 kDa) is consistent with both a ferredoxin-like [13,15] and a regulatory protein- like role in the overall PH catalyzed reaction [3,9]. The absence of redox centres (FAD, Fe/S) excludes the first hypothesis and therefore a direct involvement of PHI in electron transfer. This conclusion is consistent with the very low degree of identity between the N-terminal sequence of PHI and those of ferredoxin-like proteins belonging to other oxygenases. On the contrary, the N-terminus of PHI is identical to that of MopN from A. calcoaceticus NCIB 8250 and very similar to that of P2 from Pseudomonas CF600, both regulatory components of PHs. PHI has been found to be strictly necessary for the phenol to catechol conversion, Fig. 4. Reconstitution of PH activity in vitro in the presence of variable amounts of each component. The assay was performed with a Clark- type electrode in the presence of 1.7 m M NADH in 100 m M Mops/ NaOHbuffer,pH 7.4,at24 °C. Data were obtained with 1 m M phenol as a substrate and are corrected by subtraction of the basal oxygen consumption. PHI concentration (0.3, 0.6 and 1.2 l M , i.e. PHI/PHO ratios: 0.5, 1 and 2) was varied over a range of PHR/PHO ratios (up to 6), keeping fixed a PHO concentration of 0.6 l M . The data were fitted to Michaelis–Menten curves. Squares and dotted line: data and fitting with PHI/PHO ratio of 0.5. Triangles and continuous line: data and fitting with PHI/PHO ratio of 1. Asterisks and broken line: data and fitting with PHI/PHO ratio of 2. Fig. 5. Effect of PHI on the flavin fluorescence of the complex PHR– PHO. Dotted line: fluorescence emission spectrum of the couple PHR– PHO (2 : 1) in Hepes/NaOH buffer, pH 7.0. Solid line: fluorescence emission spectrum after the addition of PHI to the above mentioned mixture. k excitation 450 nm. Fig. 3. Temperature dependence of PHI far-UV circular dichroism spectra. Conditions: 10 l M PHI in 10 m M sodium-phosphate buffer, pH 7.0; spectra were registered at scan speed of 50 nmÆmin )1 , with 3 accumulations. The inset shows the progressive decrease of molecular ellipticity at k ¼ 200 in the temperature range of 10–70 °C. Before circular dichroism analysis, the samples were preincubated at the indicated temperatures, for 10 min, in sealed quartz cuvettes. 1438 E. Griva et al. (Eur. J. Biochem. 270) Ó FEBS 2003 as the corresponding regulatory proteins are in the reactions catalyzed by xylene monooxygenase from Pseudomonas stutzeri [14] and alkene monooxygenase from Xantobacter Py2 [15]. In other enzymes (MMO from M. capsulatus and Methylocistis [5,7], T2M from Burkolderia cepacia [4]), the regulatory protein acts as an enhancer, but it is not absolutely required for the reaction. The optimal ratio reductase: regulatory: oxygenase component, as observed in M.capsulatus MMO [27], involves equimolar concentrations (in terms of monomeric units) of the various components. Excess of PHI over the oxygenase component does not cause inhibition of the overall enzyme activity, in contrast to what observed for M. trichosporium MMO [26]. PHI coelutes with PHR in the chromatographic step that separates PHO. From the gel filtration column that separates it from PHR, PHI elutes as an 18-kDa dimer. The mechanism by which PHI activates PH is still poorly understood. One possibility is that PHI interacts with one PHR and one PHO (abc) protomer. The hypothesis of a direct PHI–phenol interaction is quite unlikely, because of the fact that the addition of phenol does not alter the emission spectrum of PHI. Moreover, PHI does not bind ANS (a probe for hydrophobic sites) and is not retained by the phenyl Sepharose column (a ligand for phenolic- recognizing sites and for hydrophobic sites in general). These results differ from those reported for the regulatory protein P2 of Pseudomonas CF600 phenol hydroxylase [9], a molecule with an N-terminus sequence very similar to that of PHI. NMR studies on P2 have suggested the presence of a hydrophobic cavity [9] that is likely to bind phenol and thus favour its interaction with the oxygenase moiety. The data here reported do not provide any evidence for the presence of a phenol-binding or other hydrophobic sites. However, we cannot exclude binding of the aromatic substrate to a buried cavity in case such an interaction would not cause changes in the protein fluorescence signal. An interaction between PHI-PHR is a likely candidate to explain the regulatory effect. In fact, on addition of PHI, the fluorescence of PHR-bound flavin increases. This finding points to a PHI-induced conformational change of PHR, possibly resulting in a more pronounced exposure of FAD to the aqueous solvent. The most important functional consequence of this PHI-induced conformational transition of PHR might be: (a) a better exposure of the Fe/S cluster involved in the electron transfer to PHO; (b) a favourable orientation of a specific PHR domain allowing for optimal interaction with PHO. If the former hypothesis were true, one could expect a more efficient electron transfer not only to PHO but also to artificial electron acceptors. However, the reduction of either cytochrome c or NBT is nearly independent of the presence of PHI. Furthermore, preliminary CV experiments do not seem to evidence any change in PHR redox potential on addition of PHI (G. Gilardi, Dept of Biological Sciences, Imperial College of Science, Technology and Medicine, London, UK, personal communication). On the other side, on the basis of X-ray scattering data, Gallagher and coworkers [11] suggested that a correct orientation of the reductase and oxygenase components of methane monooxygenase is strictly necessary to facilitate intramolecular electron transfer. PHI might similarly play the role of properly orienting the other components with respect to each other. A third mechanism of action, that has been proposed for the regulatory component of monooxygenases [28], involves its direct interaction with the oxygenase. On the basis of MCD studies, it was found that in methane monooxygenase from M.capsulatus the complexation of the regulatory component (MMOB) with the oxygenase (MMOH) induces a conformational change in the active site pocket of the MMOH a-subunit, leading to a better substrate interaction with the dinuclear iron centre [28]. This finding was confirmed by NMR spectroscopic studies, revealing that MMOB is embedded in the canyon between the two moieties of the oxygenase component (MMOH) [29]. As revealed by DPV data, the MMOH a subunit conforma- tional change-induced by MMOB, causes a decrease of the redox potential of the dinuclear iron centre [12]; further- more, EPR studies evidenced a change in M. trichosporium MMOH signal upon addition of MMOB [30]. This model is not operating in the case of PH from A. radioresistens S13, as shown by the lack of alteration in the PHO-ANS fluorescence upon addition of PHI. In summary, while the regulatory components of MMOs act via an interaction with the oxygenase [28–30], and, in the case of Pseudomonas CF600 phenol hydroxylase, via a direct interaction with the substrate itself [9], in the case of A. radioresistens S13 phenol hydroxylase, PHI appears to interact with the reductase moiety. This PHI–PHR interac- tion promotes the PHR conformational changes that are necessary to optimize the mutual orientation of PHR and PHO and thus electron transfer between them. Acknowledgements This work is supported by the EC Biotechnology programme, contract BIO-960413. We are grateful to D. Corpillo (University of Turin) for mass spectroscopy analysis, to A. Conti and G. Giuffrida (CNR- Torino) for N-terminal sequence determination and to D. Cavazzini (University of Parma) for helpful discussion and CD technical assistance. Table 1. Catalytic parameters of A. radioresistens S13 PHR, alone and in the presence of PHI, determined with two artificial electron acceptors. The K m and k cat values were determined at 30 °C, in 50 m M Tris/sulfate buffer, pH 8.5, using NADH as the electron donor. Cytochrome c NBT K m (l M ) k cat (s )1 ) k cat /K m (s )1 Æl M )1 ) K m (l M ) k cat (s )1 ) k cat /K m (s )1 Æl M )1 ) PHR 1.3 ± 0.3 61 ± 6 47 10 ± 3 0.63 ± 0.08 0.063 PHR + PHI 1.5 ± 0.2 55 ± 7 36 9 ± 3 0.66 ± 0.05 0.070 Ó FEBS 2003 A. radioresistens S13 phenol hydroxylase regulatory component (Eur. J. Biochem. 270) 1439 References 1. Pessione, E., Divari, S., Griva, E., Cavaletto, M., Rossi, G.L., Gilardi, G. & Giunta, C. (1999) Phenol hydroxylase from Acine- tobacter radioresistens is a multicomponent enzyme: purification and characterization of the reductase moiety. Eur. J. Biochem. 265, 549–555. 2. Powlowski, J. & Shingler, V. (1990) In vitro analysis and polypeptide requirements of multicomponents phenol hydroxy- lase from Pseudomonas sp. strain CF600. J. Bacteriol. 172, 6834– 6840. 3. 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