Báo cáo khoa học: Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli ppt

Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials Satoshi Hirata, Toshitaka Matsui, Yukie Sasakura, Shunpei Sugiyama, Tokiko Yoshimura, Ikuko Sagami and Toru Shimizu Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme- regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand. In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex. How- ever, the Fe(III) complexes of these mutants are six-coordi- nated low-spin, like the wild-type enzyme. The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95. To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein. CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme. The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were consid- erably lower than that of the wild-type enzyme (+70 mV) at )1, )26, and )122 mV vs. SHE, respectively. Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme. Keywords: heme sensor; magnetic circular dichroism; optical absorption; phosphodiesterase; redox potential. Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) has been cloned, and its structure–function relationships have been partially characterized by both our group and the Kitagawa group [1,2]. Interestingly, phosphodiesterase (PDE) is active only when the heme iron is in the Fe(II) redox state. Consistent with this finding, the PDE activity of Ec DOS was dramatically inhibited by CO and NO, which display strong affinities for the Fe(II) complex. Therefore, Ec DOS is likely to be a heme-based sensor, in which the redox state of the heme iron appears to control protein conformational changes, which in turn transmit signals to other domains to initiate the PDE catalytic function. The heme-bound N-terminal portion of Ec DOS has been identified as a PAS domain, based on the sequence and tertiary structure. PAS is an acronym formed from the names of proteins in which imperfect repeat sequences were initially recognized specifically: Drosophila period clock protein (PER) [3], vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT) [4], and Drosophila single- minded protein (SIM) [5]. Site-directed mutagenesis studies reveal that one of the two axial ligation sites of the heme iron is occupied by His77 [1,2]. The Gilles-Gonzalez group reported on the physicochemical properties of the isolated N-terminal heme-bound PAS domain of Ec DOS and suggested that the heme axial ligand trans to His77 is Met95 on the basis of sequence homology data and the crystal structures of other heme-bound PAS proteins [6,7]. How- ever, resonance Raman spectra of Met95Ala and Met95His mutants of Ec DOS suggest that a different molecule occupies this axial position of the Fe(III) complex [2]. Therefore, it is of interest to determine the characteristics of Met95 mutants of Ec DOS using other spectroscopic techniques and kinetic analyses. Optical absorption [8–10], magnetic circular dichroism (MCD) [10–12], and CD [13,14] spectroscopy are useful tools for examining the heme coordination structure, protein folding and the environment of aromatic amino acid residues. Here, we present an initial report on the optical absorption, MCD and CD spectra of Met95 mutants of both full-length Ec DOSandtheisolatedPAS Correspondence to T. Shimizu, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Fax: + 81 22 217 5604, 5605, 5390, Tel.: + 81 22 217 5604, 5605, E-mail: shimizu@tagen.tohoku.ac.jp Abbreviations: Ec DOS, heme-regulated phosphodiesterase obtained from Escherichia coli; PDE, phosphodiesterase; PAS, acronym formed from the names Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM); MCD, magnetic circular dichroism; FixL, oxygen sensor heme protein from Rhizobium meliloti. (Received 19 June 2003, revised 12 October 2003, accepted 16 October 2003) Eur. J. Biochem. 270, 4771–4779 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03879.x domain. Mutations at Met95 did not alter the spin state from six-coordinated low spin to five-coordinated high spin in the Fe(III) complex in Soret and visible absorption and MCD spectra, but caused significant changes in the Soret CD peak position, and dramatically decreased the redox potentials. We discuss the heme coordination structure of this redox-sensitive heme sensor, Ec DOS, in view of our spectral findings. Experimental procedures Materials and proteins The fluorescent substrate, 2¢-o-anthraniloyl adenosine 3¢,5¢- cyclic monophosphate (ant-cAMP), was purchased from Calbiochem (La Jolla, CA, USA). Calf intestine alkaline phosphatase was purchased from Takara Syuzo Co (Otsu, Japan). DEAE-Sephadex and Sephadex G25 were obtained from Amersham Biosciences (Uppsala, Sweden). Other chemicals were acquired from Wako Pure Chemicals (Osaka, Japan). Cloning, mutations, expression in E. coli and purification of full-length Ec DOS (amino acids 1–807) and the isolated heme-bound PAS domain (amino acids 1–133) were performed as described previously [1,2]. Site-directed muta- genesis was performed using the PCR-based strategy with the ODA-LA PCR kit from Takara Shuzo. The sequence was confirmed by Sanger’s method using a DSQ-2000 L automatic sequencer (Shimadzu Co.). Ec DOS-PAS mutant proteins were expressed in BL21 cells. Purified full- length Ec DOS and isolated PAS domain were more than 95% homogeneous, as confirmed by SDS/PAGE. Molar absorption coefficients of the wild-type Fe(III) and Fe(II) complexes were 129 cm )1 Æm M )1 (at 416 nm) and 175 cm )1 Æm M )1 (at 427 nm), respectively, as determined by the pyridine–hemochromogen method [18,19]. The coeffi- cients of the Met95Ala (at 414 nm), Met95Leu (at 414 nm) and Met95His (at 415 nm) mutant Fe(III) complexes were 124, 124 and 126 cm )1 Æm M )1 , respectively. Optical absorption, MCD, CD and fluorescence spectra Optical absorption experiments were performed on Shim- adzu UV-1650, UV-2500 and Hitachi U-2010 spectropho- tometers maintained at 25 °C by a temperature controller. MCD spectra were obtained on a Jasco J-500 spectro- polarimeter equipped with an electromagnet which produces a longitudinal magnetic field in the sample (up to 1.53 T). CD spectra were obtained with Jasco J-720 and Jasco J-500 CD spectropolarimeters. Fluorescence spectra were obtained using a Shimadzu RF-5300PC spectrofluorimeter. To ensure the appropriate temperature of the solution, the reaction mixture was equilibrated for 10 min before each spectroscopic measurement. Redox potentials Anaerobic spectral experiments were performed on a Shimadzu UV-160A spectrophotometer in a glove box. Redox potentials were obtained on the same spectro- meter in the glove box. 2,3,5,6-Tetramethylphenylenedi- amine (10 l M ), N-ethylphenazonium ethosulfate (10 l M ) and 2-hydroxy-1,4-naphthoquinone (10 l M ) were added as mediators to the wild-type protein solution before titration [15]. For Met95Ala and Met95Leu mutants, the dye concentrations were changed to 5 l M . For the Met95His mutant, 5 l M anthraquinone-2,6-disulfonate was added to the solution. The heme protein concentration used was 15 l M . Spectral changes in intensity at 563 nm for wild- type, 560 nm for Met95Ala, 557 nm for Met95Leu and 563 nm for Met95His accompanying the redox change were monitored, as dye absorption hampers the detection of Soret spectral changes. To ensure that the appropriate temperature of the solution was maintained, the reaction mixture was incubated for 10 min before spectroscopic measurements. For reduction titration, the sample was initially fully oxidized with potassium ferricyanide. After each addition of sodium dithionite and allowing equilibrium and stabilization, spectra were recorded. Titration experi- ments were repeated at least three times for each complex. Enzymatic assays PDE activities were measured using a fluorescence band of the product, as described previously [1,19,20]. Briefly, Ec DOSwasincubatedat37°Cwithant-cAMPina 500 lL reaction mixture comprising 50 m M Tris/HCl buffer (pH 8.5), 2 m M MgCl 2 and 1 m M dithiothreitol. To terminate the reaction, Ec DOS was removed using an Ultrafree-MC centrifugal filter (Millipore Co., Bedford, MA, USA). Then 2 U alkaline phosphatase was added to the mixture and incubated for 1 h at 37 °C. Next, the reaction mixture was applied to a DEAE-Sephadex column and washed with water. Finally, the fluorescence intensity (excitation at 330–350 nm, emission at 410 nm) of the eluted fraction was measured to determine the amount of product [1]. At least four experiments were conducted to obtain each value. Experimental errors were less than 20%. Theenzymewasreducedwith10m M sodium dithionite. Excess sodium dithionite was removed using a gel-filtration column (Sephadex G25) in a glove box under nitrogen atmosphere with an O 2 concentration of less than 50 p.p.m. [18,19]. The reaction of the Ec DOS Fe(II) complex was performed under anaerobic conditions in the glove box. Results Optical absorption spectra Optical absorption spectra (350–650 nm) of the Fe(III), Fe(II), and Fe(II)–CO complexes of wild-type and Met95 mutants of the isolated PAS domain are shown in Fig. 1 (lower), and summarized in Table 1. The absorption spectrum (peaks at 416, 530 and 564 nm) of the Fe(III) complex of wild-type Ec DOSisthatofatypicalsix- coordinated low-spin complex. This spectrum closely resembles that of cytochrome b 562 (peaks at 418, 530 and 564 nm), but shows less similarity to the spectra of other six- coordinated low-spin heme proteins, including cytochrome c and cytochrome b 5 [20–24] (Table 1). To identify the axial ligand trans to His77, we generated Met95Ala, Met95Leu and Met95His mutants. Soret and visible absorption peaks of the Fe(III) Met95 mutant proteins disclosed six-coordi- nated low-spin complexes essentially similar to that of the 4772 S. Hirata et al.(Eur. J. Biochem. 270) Ó FEBS 2003 wild-type enzyme (Fig. 1, Table 1). Notably, denaturation of the Met95 mutants occurred occasionally at high temperatures during expression or as a consequence of auto-oxidation of the O 2 -bound Fe(II) complexes during purification, often resulting in five-coordinated Fe(III) high- spin complexes. The close similarities between the absorption spectra of wild-type Ec DOS and cytochrome b 562 are also evident in the Fe(II) complexes (Table 1). Absorption spectra of Fe(II) complexes of the Met95Ala and Met95Leu mutants were characteristic of five-coordinated high-spin complexes sim- ilar to Fe(II) myoglobin, whereas the spectrum of the Fe(II) complex of the Met95His mutant was characteristic of a six-coordinated low-spin complex, analogous to the Fe(II) complex of the wild-type enzyme. Absorption spectra of Fe(II)–CO complexes of the Met95 mutants were compar- able to that of the wild-type enzyme and very similar to the spectrum of the corresponding Fe(II)–CO myoglobin complex (Table 1). Spectra generated using mutants of full-length Ec DOS were essentially identical with those of the isolated PAS domain. To identify axial ligand(s), the effect of modulating pH on absorption bands was examined using the isolated PAS domain, as holoenzymes are easily precipitated when pH is varied. Between pH 3.5 and 9, no spectral changes were observed for either the wild-type or Met95His mutant PAS domain. However, Met95Ala and Met95Leu mutants form six-coordinated high-spin complexes between pH 4 and 7.5. The Met95Leu mutant is likely to have a pK a at  4.6 (data not shown), but partial denaturation occurs concomitantly with this spin-state shift in the acidic region. The Met95Ala mutant was easily denatured when pH was varied below 6, andnoclearpKa value was obtained. MCD spectra Figure 1 (upper) displays the MCD spectra of Fe(III), Fe(II) and Fe(II)–CO complexes of wild-type (A), Met95Ala (B), Met95Leu (C) and Met95His (D) mutants of Ec DOS. MCD positions and intensities are summarized in Table 2. The MCD spectrum of the Fe(III) complex of the wild-type enzyme contained a peak at 412 nm and a trough at 426 nm in the Soret region, and a small peak at 554 nm and a small trough at 569 nm in the visible region (solid line in Fig. 1A). The MCD spectral contour of the wild-type enzyme Fe(III) complex is typical of a Fig. 1. Optical absorption (lower panel) Soret and visible MCD (upper panel) spectra for the Fe(III) (—), Fe(II) (- - -), and Fe(II)-CO ( … ) complexes of wild-type (A), Met95Ala (B), Met95Leu (C) and Met95His (D) mutants of isolated heme-bound PAS domain. MCD spectra of the wild-type and Met95 mutants of the full-length enzyme were similar to those of the isolated heme-bound PAS domain. Spec- tra were obtained in 0.1 M Tris/HCl (pH 8.0) buffer. Molar absorption coefficients deter- mined are described in Experimental pro- cedures. Ó FEBS 2003 Met95 mutants of heme-regulated phosphodiesterase (Eur. J. Biochem. 270) 4773 six-coordinated low-spin complex. The intensity (De/T) of the Soret trough band  80 M )1 Æcm )1 ÆT )1 is also typical of a low-spin complex [11]. Interestingly, the Met95Ala, Met95Leu and Met95His mutants of Ec DOS displayed similar MCD spectra (solid lines in Fig. 1B–D) to the wild- type enzyme, although a specific decrease in Soret MCD intensity was observed for the Met95Ala and Met95Leu mutants (Table 2). Note that denaturation occurring during the purification procedures often generated five-coordinated high-spin Fe(III) complexes for the Met95 mutants, inclu- ding Met95Phe (not shown). The Fe(II) complex of the wild-type enzyme had a peak at  430 nm in the Soret region at a comparable intensity to the Fe(III) complex (Fig. 1A, broken line). MCD intensities recorded in the visible region for the wild-type enzyme are more than twofold higher than those in the Soret region, unlike those of the Fe(III) complex. A similar MCD pattern was observed for the Met95His mutant (Fig. 1D). However, MCD band intensities in the visible spectra of the Met95Ala and Met95Leu mutant proteins were much lower than those in the Soret region. The position of the MCD peak (437 nm) in the Soret region of Met95Ala, Met95Leu and Met95His mutants was higher than that (430 nm) of the wild-type protein. Moreover, the Soret MCD spectra of the wild-type enzyme and the Met95His mutant started on the plus side and switched to the minus side (from lower to higher wavelengths), which was reversed for Met95Ala and Met95Leu mutants. The MCD data indicate that the Fe(II) complexes of the wild-type and Met95His mutant are characteristic of spectra of the low-spin complexes in that intensities of the visible MCD bands are much higher than those of the Soret MCD bands [10,11]. On the other hand, the Met95Ala and Met95Leu mutant complexes are high- spin, as the MCD intensities on the plus side in the Soret region are much higher than those in the visible region [10,11]. The Soret MCD peaks of the Fe(II)–CO complexes of the wild-type and Met95 mutants were located at  420 or 421 nm, with troughs at  430 or 431 nm (Fig. 1 and Table 2, dotted lines). The MCD peaks and troughs in the visible region of these proteins were observed at  562– 564 nm and 579–580 nm, respectively. However, the MCD spectra of the Fe(II)–CO complexes of the wild-type and Met95His mutant proteins differed from those of the Met95Ala and Met95Leu mutants. Specifically, the Soret MCD intensities of the Fe(II)–CO complexes of the Met95Ala and Met95Leu mutants were 20–30% higher than those of the Fe(II)–CO complexes of the wild-type enzyme and Met95His mutant (Table 2). Soret CD spectra The a-helix and b-sheet contents of the full-length enzyme andisolatedPASdomainweredeterminedfromtheUV region of the CD spectrum in a previous study by our group [1]. Neither reduction of the heme iron nor Met95 mutations altered the CD spectrum in this region (data not shown). Soret CD bands of the proteins under various conditions are shown in Fig. 2 and summarized in Table 3. The Soret CD band of the Fe(III) wild-type complex was presented at  421 nm on the plus side with De ¼ 26.2 M )1 Æcm )1 (solid line in Fig. 2A). Reduction with sodium dithionite led to a shift in the band position to 431 nm and a slight increase in CD intensity (up to De ¼ 32.3 M )1 Æcm )1 : broken line in Table 1. Optical absorption maxima (nm) of the wild-type and Met95 mutants of isolated Ec DOS PAS domain. Putative coordination structures are specified in parentheses. Optical absorption wavelengths of full-length enzymes were similar to those of the isolated PAS domain. SwMb, Sperm whale myoglobin. Proteins Fe(III) Fe(II) Fe(II)–CO Reference Wild-type 416, 530, 564 (6c-LS) 427, 532, 563 (6c-LS) 423, 540, 570 (6c-LS) This work Met95Ala 414, 534, 563 (6c-LS) 432, 560 (5c-HS) 423, 541, 571 (6c-LS) This work Met95Leu 414, 533, 561 (6c-LS) 434, 558 (5c-HS) 423, 541, 570 (6c-LS) This work Met95His 415, 533, 564 (6c-LS) 427, 532, 561 (6c-LS) 423, 541, 570 (6c-LS) This work His, Met: axial ligands (6c-LS) Cyt c 408, 530, 695 413, 521, 550 [20] Cyt c 2 416, 550 [21] Cyt c 551 410, 520, 551 416, 520, 551 [22] Cyt b 562 418, 530, 564 427, 531, 562 [23] His, His: axial ligands (6c-LS) Cyt c 7 408 418, 522, 552 [24] Cyt b 5 413, 540 (br) 423, 526, 556 [23] His: axial ligand SwMb (H 2 O) 410, 505, 635 (6c-HS) 434, 556 (5c-HS) 423, 542, 579 (6c-LS) [8] (OH – ) 414, 542, 582 (6c-LS) [8] 4774 S. Hirata et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 2A). The Fe(II)–CO complex displayed a Soret band at  425 nm on the plus side with high intensity (De ¼ 58.5 M )1 Æcm )1 : dotted line in Fig. 2A). Soret CD bands of the Fe(III) complexes of all the Met95 mutants were observed at  414 nm, in contrast with that of the wild-type enzyme complex, which was at  421 nm. The distinct Soret CD positions of the Fe(III) complexes of the wild-type enzyme and the Met95 mutants is important, in view of the fact that the optical absorption, MCD, and resonance Raman spectra of the wild-type and mutant proteins are essentially similar [1,2]. The Soret CD bands of the Fe(II) Met95Ala and Met95Leu mutants were presented at  435–437 nm, at a slightly higher position than those of the wild-type enzyme and Met95His mutant (430–431 nm). Redox potentials Electrochemical titrations were conducted for the Met95 mutants of the isolated PAS domain, as full-length enzymes precipitate in the presence of mediators. The one-electron midpoint potential of the Ec DOS heme was determined from the absorbance change at 560 nm (Fig. 3). Redox potentials obtained for the Met95 mutants ()1to)122 mV vs. SHE) summarized in Table 4 are much lower than that of the wild-type enzyme (+70 mV). Oxidative titrations were also conducted for the Met95 mutant proteins to determine whether a redox-dependent ligand switch occurs. No significant differences were observed between the mid- point potentials of reductive and oxidative titrations. Interestingly, wild-type Ec DOS has a positive potential, similartohemeproteinsinvolvedinelectrontransferorO 2 storage, while Met95 mutants displayed lower, negative potentials similar to the enzymes involved in O 2 or H 2 O 2 activation, such as cytochromes P450 and peroxidases (Table 4). Table 2. MCD spectra of wild-type and Met95 mutants of isolated Ec DOS PAS domain. Intensities (indicated in parentheses) are expressed as De/H ( M )1 Æcm )1 ÆT )1 ). MCD peaks and intensities of full- length enzymes were similar to those of the isolated PAS domain. Soret Visible Peak Crossover Trough Peak Crossover Trough Fe(III) Wild-type 412 (78.9) 420 426 ()61.4) 554 (12.3) 563 569 ()9.8) Met95Ala 410 (68.1) 418 425 ()55.2) 556 (8.7) 567 580 ()7.5) Met95Leu 410 (65.0) 418 425 ()48.9) 556 (8.9) 571 580 ()10.2) Met95His 410 (71.5) 417 425 ()67.7) 5.54 (9.9) 563 571 ()8.3) Fe(II) Wild-type 430 (70.9) 558 (176) 561 565 ()179) Met95Ala 437 (94.5) Met95Leu 437 (109) Met95His 436 (36.1) 555 (95.9) 559 563 ()97.7) Fe(II)–CO Wild-type 420 (68.9) 427 431 ()26.9) 564 (18.4) 571 580 ()25.1) Met95Ala 421 (88.6) 427 431 ()31.7) 563 (21.8) 571 579 ()24.9) Met95Leu 420 (92.1) 426 430 ()36.2) 563 (25.0) 570 579 ()28.1) Met95His 420 (73.6) 427 431 ()27.4) 562 (19.2) 570 579 ()22.6) Fig. 2. Soret CD spectra of the Fe(III) (—), Fe(II) (- - -), and Fe(II)-CO ( … ) complexes of wild-type (A), Met95Ala (B), Met95Leu (C) and Met95His (D) mutants of isolated heme- bound PAS domain. CD spectra of wild-type and Met95 mutants of the full-length enzyme were similar to those of the isolated heme-bound PAS domain. Concentrations are 7.6 l M for wild-type, 5.8 l M for the Met95Ala mutant, 4.5 l M for the Met95Leu mutant, and 5.6 l M for the Met95His mutant in 0.1 M Tris/HCl (pH 8.0) buffer. Ó FEBS 2003 Met95 mutants of heme-regulated phosphodiesterase (Eur. J. Biochem. 270) 4775 cAMP PDE activities Wild-type Ec DOS displays PDE activity only in the Fe(II) form [1]. PDE activities of Fe(II) Met95 mutants of the holoenzyme were obtained under anaerobic conditions. Interestingly, all Met95 mutant proteins displayed PDE activities that were comparable to that of the wild-type enzyme. In addition, the Fe(III) Met95 mutants exhibited no PDE activity, similar to the wild-type enzyme. Our data clearly demonstrate that Met95 is not essential for PDE activity with cAMP. Discussion Optical absorption and MCD spectra A comparison of the absorption spectra of Fe(III) and Fe(II) Ec DOS proteins with those of the corresponding six- coordinated low-spin complexes of other heme proteins reveals very similar spectral patterns between Ec DOS proteins and cytochrome b 562 (Table 1). Although cyto- chrome b 562 contains His/Met axial ligands, the spectra are distinct from those of other cytochromes with His/Met or His/His ligation. Therefore, the coordination structure and/or heme environment of Ec DOS appears similar to that of cytochrome b 562 , if the axial ligands of the two heme proteins are the same. Our previous data indicate that His77 is one of the axial ligands of the heme in Ec DOS [1,2]. Amino-acid sequences and crystal structures of PAS proteins indicate that the axial ligand trans to His77 is possibly Met95 [6,7,31]. Construc- tion of the distal heme site of Ec DOS by replacing the Ec DOS sequences on the FixL backbone indicates that Met95 is possibly the axial ligand trans to His77, based on the finding that there are no other amino acid(s) in a suitable position to fulfil this role, except for coordination of a water molecule or a main chain amide. However, there remains another possibility, that the water molecule or hydroxy anionthatinteractswithMet95isanaxialligandtothe Fe(III) heme. The Fe(III) complexes of all three Met95 mutants displayed the same coordination structure as the wild-type enzyme (six-coordinated low-spin in terms of the Soret and visible optical absorption and MCD spectra) (Tables 1 and 2). Resonance Raman spectra of the Met95Ala mutant in addition disclosed that the Fe(III) form of this mutant is six- coordinated low-spin [2]. If Met95 is an axial ligand to the heme, the Fe(III) forms of the Met95Ala and Met95Leu mutants should display five-coordinated high-spin states, because Ala and Leu have nonpolar aliphatic side chains and cannot coordinate to the heme. Changes dependent on pH were observed in the absorption spectra of Met95Ala and Met95Leu mutants, although the Met95Ala mutant was denatured to some extent at acidic pH. It is possible that ahydroxyaniontrans to His77 in the six-coordinated low- spin complex is protonated, resulting in a five-coordinated high-spin complex bound by His77 with the release of water. Table 3. Soret CD spectra of wild-type and Met95 mutants of the iso- lated Ec DOS PAS domain. Intensities are expressed as De ( M )1 Æcm )1 ). CD peaks and intensities of full-length enzymes were similar to those of the isolated PAS domain. Note that CD intensities and band positions for the Fe(II) and Fe(II)–CO complexes in [1] are incorrect. Peak (nm) Intensity Fe(III) Wild-type 421 26.2 Met95Ala 414 28.3 Met95Leu 414 27.0 Met95His 414 27.6 Fe(II) Wild-type 431 32.3 Met95Ala 437 30.1 Met95Leu 435 37.6 Met95His 430 28.8 Fe(II)–CO Wild-type 425 58.5 Met95Ala 423 61.7 Met95Leu 423 70.7 Met95His 423 56.9 Fig. 3. Reductive titrations of the isolated Ec DOS PAS domain wild- type (d), the Met95Leu (n), Met95Ala (h)andMet95His(s)mutants from right to left. The fraction of the ferrous form was plotted as a function of the potential. Solid lines represent theoretical Nernst curves for the reduction of wild-type and Met95 mutants. Oxidative titrations did not lead to any significant shifts. Table 4. Redox potentials of wild-type and Met95 mutants of the isolated Ec DOS PAS domain. Protein mV vs. SHE n Reference Wild-type +70 or +67 0.95 This work, [1] Met95Leu )1 0.98 This work Met95Ala )26 0.98 This work Met95His )122 0.96 This work Cytochrome c +260 [16] Cytochrome b 562 +140 (pH 8.5) [23] Myoglobin +59 or +46 [25–27] Cytochrome b 5 +3 or +20 [23,28] Cytochrome P450cam )170 (+ substrate) [27,29] 303 (– substrate) [27,29] Horseradish peroxidase )220 or – 270 [26,27] Microsomal cytochrome P450 )310 [30] 4776 S. Hirata et al.(Eur. J. Biochem. 270) Ó FEBS 2003 However, the apparent pK a of 4.6 is unusually low for a transition between an hydroxy anion and water. This suggests significant structural differences in the heme environments of the wild-type enzyme and the Met95Ala and Met95Leu mutants. It is possible that an unknown ligand, such as water/hydroxylate anion (or an amino-acid main chain), coordinates to the Fe(III) in Met95Ala and Met95Leu mutants to produce low-spin complexes. This type of coordination may occur because of close proximity of the water/hydroxyl to Met95 or flexibility of the environment on the heme distal side. For the Fe(II) complexes, optical absorption spectra of the Met95 mutants are consistent with predictions from the amino-acid sequences and crystal structures of the PAS proteins. Specifically, the Met95Ala and Met95Leu mutants form five-coordinated high-spin complexes, whereas the complex of the Met95His mutant is six- coordinated low-spin, similar to that of the wild-type enzyme, indicating that His is the sixth axial ligand in the Fe(II) Met95His complex. Optical absorption spectra of the Fe(II)–CO complexes of the wild-type and Met95 mutants are similar, indicating the presence a common internal axial ligand, His77, as expected from the resonance Raman spectra of the proteins [2]. The spin and electronic states determined from the MCD spectra of the Met95 mutants analyzed in this study are consistent with those obtained from the optical absorption spectra. The MCD spectra of the Met95 mutants indicate that substitution with Ala, Leu and His did not alter the spin state of the Fe(III) complex, whereas Met95Ala and Met95Leu mutations changed the spin state of the Fe(II) complex from low spin to high spin. However, the Soret MCD intensities of the Fe(II)–CO complexes of the Met95Ala and Met95Leu mutants were higher than those of the wild-type enzyme and Met95His mutant. It appears that ligand displacement accompanies the Met95 mutations, andMet95maybethesixthaxialligandintheFe(II) complex. Therefore, it suggests that redox-dependent axial ligand exchange occurs, as indicated by resonance Raman spectroscopy [2]. Soret CD spectra The Soret CD band of the Fe(III) form of Ec DOS is located exclusively on the plus side. This is distinct from cytochrome b 562 [32] and cytochrome c [33] complexes, which display bands on the minus side and both sides, respectively. The Soret CD band of Ec DOS shifted from 421 nm to 425 nm and the intensity increased on reduction, suggesting a change in the heme environment. The origin of the Soret CD band of the heme protein may be due to interactions of the p to p* transition of nearby aromatic side chains with the delocalized p-electron system of the heme prosthetic group and/or the unique conformation of the polypeptide surrounding the heme [13,33–35]. In fact, Phe mutations markedly altered the Soret CD band shape of cytochrome c [33]. Importantly, the Soret CD positions of all the Fe(III) Met95 mutants are 7 nm lower in wavelength than that of the wild-type enzyme. The Soret CD spectra of the Fe(III) Met95 mutants thus indicate that substitutions at this position result in similar and profound conformation changes near the heme prosthetic group in this particular oxidation state. Therefore, it is reasonable to speculate that the Met95 mutations affect the 6th ligand (water/hydroxy ion) of the Fe(III) complex at the heme distal side. From the Soret CD bands of the Fe(II) complexes, it appears that the heme environment structures of the Fe(II) wild-type enzyme and the Met95His mutant are distinct from those of the Fe(II) Met95Ala and Met95Leu mutants. These observations appear consistent with the optical absorption, MCD and resonance Raman spectra, which suggest that the Fe(II) wild-type and Met95His mutant are in the low-spin state, whereas the Fe(II) Met95Ala and Met95Leu mutants are high-spin [1,2]. The Soret CD band positions and intensities of the Fe(II)–CO complexes of the wild-type and Met95 mutants are similar. Redox potentials If a redox-dependent ligand switch occurs, the value for the oxidative titration would be expected to be different from that for the reductive titration. However, no such difference was observed in the two directions. However, if a redox- dependent ligand switch occurs and one of the axial ligands in either redox state is a water molecule or hydroxy anion, it is possible that the values for the two directions are similar, because the ligand switching does not need high energy. The redox potential of the wild-type protein was +70 mV vs. SHE, which is within the range of electron-transfer hemoproteins, cytochromes, and myoglo- bin (Table 4). On mutation of Met95 to Ala, Leu and His, the redox potential was markedly decreased in all the mutants studied (Table 4), suggesting that the mutations stabilize the Fe(III) form rather than the Fe(II) form. Optical absorption and MCD spectral data are consistent with the theory that Met coordination favours the Fe(II) form, whereas His coordination favors the Fe(III) form. Again, it is implied that Met95 is not an axial ligand in the Fe(III) complex. The redox potentials of the Met95 mutants are negative, similar to cytochrome P450 enzymes and peroxidases. On the other hand, redox potentials of cytochromes and myoglobin are positive (Table 4). There- fore, Met95 of Ec DOS appears to be an important redox potential control residue, preventing the enzyme from exhibiting peroxidase-like and/or P450-like behavior and avoiding activation of molecular oxygen or H 2 O 2 under aerobic conditions. Catalytic activities All Met95 mutants are active only when the heme iron is in the Fe(II) state, similar to the wild-type Ec DOS enzyme [1]. The Fe(II) complexes of Met95Ala and Met95Leu mutants are five-coordinated high-spin, in contrast with the Fe(II) form of the wild-type enzyme, which is six-coordinated low- spin. From the spectra, it appears that the axial ligand trans to His77 is Met95 in the Fe(II) form [2]. Therefore, the changes in the Fe(II) heme coordination structure and electronic state caused by the Met95 mutations did not greatly influence the PDE activity of Ec DOS. These structural changes are induced on the heme distal side, where CO and O 2 are expected to bind. Ec DOS activity is sensitive to the heme oxidation state. Accordingly, we Ó FEBS 2003 Met95 mutants of heme-regulated phosphodiesterase (Eur. J. Biochem. 270) 4777 speculate that the heme proximal site structure (including the Fe-His N distance and/or conformation), which alters depending on the oxidation state, is critical for regulating the PDE activity of Ec DOS. Detailed structural studies on the environment near the heme are required to explain the redox-sensitive PDE activity of this enzyme. Crystal structure After we submitted this manuscript, crystal structures of the isolated heme-bound PAS domain of the wild-type Ec DOS protein (resolution 1.3 A ˚ , R ¼ 0.16) were determined in this laboratory (H. Kurokawa, unpublished results). The struc- tures indicate that the axial ligands of the Fe(III) complex are His77 and water, whereas those of the Fe(II) complex are His77 and Met95. The redox-dependent ligand switch suggested from the solution and crystallographic studies of the enzyme agree well with each other, and this fact increases the value of both approaches. Thus, artifacts resulting from amino-acid substitution, experimental con- ditions, data misinterpretation or the crystallization process can be ruled out in the 3D structure determination work. Acknowledgements We thank Dr Hirofumi Kurokawa for valuable suggestions. References 1. 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The MCD data indicate that the Fe(II) complexes of the wild-type and Met95His mutant are characteristic of spectra of