Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme- regulated phosphodiesterase from Escherichia coli Nao Yokota, Yasuyuki Araki, Hirofumi Kurokawa, Osamu Ito, Jotaro Igarashi and Toru Shimizu Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan The heme-regulated phosphodiesterase from Escheri- chia coli (Ec DOS) is a heme-based sensor enzyme composed of two functional domains: an N-terminal domain with a PAS structure that contains the heme iron; a C-terminal domain that contains the phospho- diesterase catalytic domain [1–8]. Ec DOS hydrolyzes cAMP when the heme iron is in the ferrous [Fe(II)] state, whereas it is inactive when the heme iron is in the ferric state [Fe(III)] [2,4,7]. Determination of the X-ray crystal structure resolved some aspects of how changes in the N-terminal sensor domain are intra- molecularly transduced to regulation of the catalytic domain [6]. Specifically, the X-ray crystal structure of the isolated heme-bound PAS domain (Ec DosH) Keywords auto-oxidation; CO binding; heme-sensor protein; O 2 binding; phosphodiesterase Correspondence 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, 5390 Tel: +81 22 217 5604, 5605 E-mail: shimizu@tagen.tohoku.ac.jp (Received 13 December 2005, revised 14 January 2006, accepted 18 January 2006) doi:10.1111/j.1742-4658.2006.05145.x The heme-regulated phosphodiesterase from Escherichia coli (Ec DOS), which is a heme redox-dependent enzyme, is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence of Ec DOS activity on the redox state. Axial ligands such as CO, NO, and O 2 act as inhibitors of Ec DOS because they interact with the ferrous heme complex. The X-ray crystal structure of the isolated heme-bound domain (Ec DosH) shows that Leu99, Phe113 and Leu115 indirectly and directly form a hydrophobic triad on the heme plane and that they should be located at or near the ligand access channel of the heme iron. We generated L99T, L99F, L115T, and L115F mutants of Ec DosH and examined their physicochemical characteristics, including auto-oxidation rates, O 2 and CO binding kinetics, and redox potentials. The Fe(III) complex of the L115F mutant was unstable and had a Soret absorption spectrum located 5 nm lower than those of the wild-type and other mutants. Auto-oxidation rates of the mutants (0.049–0.33 min )1 ) were much higher than that of the wild- type (0.0063 min )1 ). Furthermore, the redox potentials of the former three mutants (23.1–34.6 mV versus SHE) were also significantly lower than that of the wild-type (63.9 mV versus SHE). Interaction between O 2 and the L99F mutant was different from that in the wild-type, whereas CO binding rates of the mutants were similar to those of the wild-type. Thus, it appears that Leu99 and Leu115 are critical for determining the characteristics of heme iron. Finally, we discuss the roles of these amino-acid residues in the heme electronic states. Abbreviations BjFixL, oxygen sensor heme protein from Bradyrhizobium japonicum; Ec DOS, a heme-regulated phosphodiesterase of Escherichia coli; Ec DosH, the isolated heme-bound PAS domain of Ec DOS; SHE, standard hydrogen electrode; SmFixL, oxygen sensor heme protein from Sinorhizobium meliloti. 1210 FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS indicates that global structural changes accompany the redox change. Specifically, there is a switch in the heme axial ligand and changes in the flexibility of the FG loop (residues 86–96) in the protein when the redox state of the heme iron is changed [6]. For the Fe(III) heme, the axial ligands are His77 and hydroxide anion, whereas those for the Fe(II) heme are His77 and Met95. In addition, the FG loop is very flexible and disordered and could not be resolved in the crystal structural for the Fe(III) heme complex, whereas the loop was rigid and could be resolved for the Fe(II) heme complex. It is expected that these structural changes in the heme-bound PAS domain are related to intramolecular signal transduction to the catalytic domain. Interestingly, CO and NO bind to the Fe(II) heme complex, inactivating the enzyme [2]. O 2 also binds to the Fe(II) heme complex and easily oxidizes the heme iron to the Fe(III) heme complex, terminating cata- lysis. Therefore, these gases should act as inhibitors by axially coordinating to the Fe(II) heme complex. Met95 is the axial ligand of the Fe(II) heme complex (Fig. 1A). Mutations at Met95 of Ec DosH markedly change the kinetic parameters for CO and O 2 binding to the Fe(II) heme complex as well as the redox poten- tial of the heme iron [5,8]. The rate of CN binding to the Fe(III) heme complex of Ec DosH is also remark- ably accelerated by the M95A and M95L mutations by 8–11-fold [9]. The crystal structure (Fig. 1B) of the Fe(II)–O 2 com- plex of Ec DosH indicates that Arg97 is hydrogen- bonded to the molecular oxygen on the heme plane [10]. A hydrophobic triad observed for other cor- responding heme-bound PAS enzymes, oxygen sensor heme protein from Bradyrhizobium japonicum (BjFixL) and oxygen sensor heme protein from Sinorhizobium meliloti (SmFix), is also observed for Ec DOS. Although the triad is composed of Ile215 (BjFixL) ⁄ Ile209 (SmFixL), Leu236 (BjFixL) ⁄ Leu230 (SmFixL), and Ile238 (BjFixL) ⁄ Val232 (SmFixL) for BjFixL and SmFixL, there is no amino acid that spatially corres- ponds to Ile215 (BjFixL) ⁄ Ile209 (SmFixL) in Ec DosH (Figs 1 and 2) [10]. Phe113 and Leu115 of Ec DosH correspond to two other members, Leu236 (BjFixL) ⁄ Leu230 (SmFixL) and Ile238 (BjFixL) ⁄ Val232 (SmFixL), respectively, of the hydrophobic triad. Leu99 serves as a third hydrophobic heme contact in Ec DosH [10]. The corresponding amino acids at posi- tion 99 of Ec DOS for the two other heme-bound PAS proteins, BjFixL and SmFxL, are Gly225 and Gly218, respectively (Fig. 2). It is well known that CO-binding and O 2 -binding access channels of myoglobin and he- moglobin are composed of hydrophobic amino acid residues [11]. The hydrophobic characteristics of these axial lig- ands facilitate their binding to the hydrophobic pocket on the heme distal side. Although the heme distal structure of the heme-bound PAS domain for Ec DOS AB Fig. 1. Structure of the heme distal side of (A) the Fe(II) (PDB code 1V9Z) and (B) the Fe(II)–O 2 (PDB code 1VB6) complexes of Ec DosH ([6,10]; our unpublished data). In (B), Met95 [yellow in (A)] is omitted to clearly illustrate the binding of O 2 to Arg97 [blue in (A) and (B)]. Figures were obtained by MOLFEAT version 2.1 (Fiatlux, Tokyo). N. Yokota et al. Heme electronic states of Leu mutants of Ec DOS FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS 1211 is different from that of myoglobin and hemoglobin (which have a globin fold), it has been speculated that the hydrophobic characteristic of the heme distal side contributes substantially to the ligand binding kinetics of the PAS domain of Ec DOS, as it does in myoglo- bin and hemoglobin. Examination of the effects of these hydrophobic amino acids on the kinetics of O 2 and CO binding to the Ec DosH heme is important because both diatomic molecules act as inhibitors of Ec DOS. Therefore, we expected that it would be worth while to determine how one (Leu115) of the hydrophobic amino acids that directly participates in the hydro- phobic triad and another (Leu99) in the second hydro- phobic contact contribute to the physicochemical characteristics of Ec DOS. We generated L99T, L99F, L115T, and L115F mutants of Ec DosH to understand how these hydrophobic amino acids contribute to O 2 and CO binding kinetics and other physicochemical characteristics such as auto-oxidation and the redox potential of the heme iron. We found that mutation of these hydrophobic amino acids substantially influences the rate of auto-oxidation and the redox potential of the heme iron, but, surprisingly, they had less effect on the characteristics of O 2 and CO binding. Finally, we discuss the roles of these hydrophobic amino acids in the structure surrounding heme and the electronic states of heme. Results and Discussion Optical absorption spectra of the Fe(III)–CO, Fe(II)–CO, and Fe(II)–CO complexes of the Ec DosH mutants The optical absorption spectra of the Fe(III)–CO, Fe(II)–CO, and Fe(II)–CO complexes of the Leu99 and Leu115 mutants of Ec DosH were essentially the same as those of the wild-type protein, except for the L115F mutant (Fig. 3, Table 1). It is thought that these mutations (except for L115F) did not alter the structure of the heme surroundings, including the heme co-ordination structure. The Fe(III) complex of the L115F mutant has the Soret absorption at 413 nm, which is lower than those of other proteins (417– 418 nm). It has been suggested that the Fe(III) com- plexes of most of the mutant proteins are in a low-spin state with His77 and hydroxide anion as the axial lig- ands, as in the wild-type protein [3,5,6,8]. However, it appears that the L115F mutant contains a different axial ligand trans to His77. Introducing a group with a large side chain, i.e. the phenyl group in L115F, may have substantially changed the heme distal side struc- ture and led to the changes in the heme co-ordination structure and ⁄ or movement of the heme plane such as sliding, twisting, or doming. When the distal axial lig- and of myoglobin was changed from OH – to water or acetate anion, the Soret peak position moved to a lower wavelength by 4–5 nm [11]. Thus, it seems that the axial ligand of the Fe(III) complex of Ec DosH switched from OH – to the water molecule as a result of the L115F mutation. The Fe(II) complexes of all of the mutant proteins in this study should have His77 and Met95 as axial lig- ands, whereas the Fe(II)–CO complexes should have CO and His77 as the axial ligands, as in the wild-type. This suggests that the structures surrounding heme, including the heme coordination structure, are essen- tially the same in the wild-type protein and the mutants generated here, except for the L115F mutant. The over-expression efficiency of the L115F mutant protein in the bacteria was comparable to that of the wild-type protein, but the heme-bound L115F mutant protein was more difficult to purify than the wild-type and other mutant proteins. Purification yield of the heme-bound L115F mutant was low, less than 10% of other proteins. The heme content of the L115F mutant was % 30%, which is significantly lower than those (60–70%) of the wild-type and other mutant proteins, suggesting that the L115F mutant has a lower ability to bind heme than the wild-type and the other mutant proteins. The L115F mutant of Ec DosH was not used for further determination of the physicochemical char- acteristics, such as the kinetics of O 2 or CO binding or Fig. 2. Partial sequences of amino acids of the PAS domains of Ec DOS and other related heme-bound PAS proteins. The bold amino acids represent those discussed in the text. Heme electronic states of Leu mutants of Ec DOS N. Yokota et al. 1212 FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS the redox potential (Tables 3, 4, 5), because strong heme binding and stability of the heme-bound protein are needed to obtain precise values. The Fe(II)–O 2 complexes and auto-oxidation We attempted to obtain spectra of the Fe(II)–O 2 heme complexes of the Ec DosH mutant proteins. The spec- tral maxima of the Fe(II)–O 2 complexes of all mutants except the L99F mutant were located at 417 nm, essen- tially the same as that of the wild-type protein (Fig. 4 and Table 2). However, we could not obtain the opti- cal absorption spectrum of the stable Fe(II)–O 2 com- plex of the L99F mutant. A similar result was also obtained for the R220I mutant of BjFixL [19]. The rates of auto-oxidation for the Ec DosH mutant proteins generated in this study were more than eight- fold higher than that of the wild-type protein (Fig. 4C, Table 2). In earlier studies, Ala and Leu substitut- ions at Met95, an axial ligand in the Fe(II) complex, Table 1. Optical absorption maxima (nm) and millimolar absorption coefficients (mM )1 Æcm )1 ) of the wild-type and mutant proteins of Ec DosH. The millimolar absorption coefficients (shown in parentheses) were determined using the pyridine hemochromogen method [26]. Fe(III) Fe(II) Fe(II)–CO Soret baSoret baSoret ba Wild-type 418 (110) 529 565 428 (149) 533 563 424 (149) 541 571 L99T 417 (112) 527 565 428 (158) 532 564 423 (178) 541 574 L99F 418 (112) 530 565 429 (160) 532 563 425 (207) 543 574 L115T 418 (105) 533 566 428 (137) 531 562 423 (168) 540 570 L115F 413 (117) 540 429 (141) 535 563 423 (240) 541 572 A C B D Fig. 3. Optical absorption spectra of Fe(III) (black), Fe(II) (red), and Fe(II)–CO (blue) complexes of the mutant proteins of Ec DosH. (A) L99T (7.0 l M per heme) (B) L99F (5.4 lM per heme) (C) L115T (7.8 lM per heme) and (D) L115F (3.4 lM per heme). N. Yokota et al. Heme electronic states of Leu mutants of Ec DOS FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS 1213 markedly decreased the rate of auto-oxidation [5], whereas Ala and Asn substitutions at Asp40, an amino-acid residue that interacts via two water mole- cules with the proximal ligand His77, markedly increased the rate of auto-oxidation [13] (Table 2). The rates of auto-oxidation appear to be influenced by the hydrogen-bonding network to O 2 , the polarity of the heme distal side, the direction of the heme-bound O 2 molecule, and the redox potential of the heme iron [5]. The reason why the auto-oxidation rate of the Leu99 and Leu115 mutants was much higher than that of the wild-type protein is currently uncertain. It is clear, however, that the polarity of the O 2 -binding site changed for the L99T and L115T mutants, whereas the stronger hydrophobic contact, strain, or compact- ness (or some combination thereof) of the O 2 -bound space may have contributed to the increase in the rates of auto-oxidation for the L99F and L115F mutants. O 2 binding kinetics The O 2 binding kinetics of the L99T, L99F, and L115T mutants was examined by using a stopped-flow spectrometer under anaerobic conditions (Fig. 5 and Table 3). The spectral changes accompanying O 2 bind- ing monitored at 429 nm were composed of two phases (% 1 : 1 ratio) for the wild-type and the L99T, L99F, and L115T mutant proteins. Both the fast and slow phases were dependent on the concentration of O 2 for all proteins except the L99F mutant, for which the slow phase was independent of the O 2 concentration. The rate constants for the fast phase of O 2 binding to the three mutant proteins [(49–75) · 10 )3 lm )1 Æs )1 ] were comparable to those of the wild-type protein [(31–81) · 10 )3 lm )1 Æs )1 ] [5]. Similarly, those rates for the slow phase of O 2 binding to the two mutant proteins [(6.8–7.2) · 10 )3 lm )1 Æs )1 ] were comparable to that for the wild-type protein (8.3 · 10 )3 lm )1 Æs )1 ) (Table 3). Therefore, it appears that mutations of Leu99 and Leu115, except for the L115F mutation, had little effect on the kinetics of O 2 binding. In our previous report, we observed only the fast phase [5], whereas only the slow phase was observed by others [1], leading to conflicting results. In this study, we used the new stopped-flow spectrometer A B Fig. 4. (A) Optical absorption spectrum of the Fe(II)–O 2 complex of the L99T mutant (7.1 l M per heme). Arrows designate spectral changes from the Fe(II)–O 2 complex (black) to the Fe(III) complex (red). (B) Time-dependent changes in intensity at 580 nm accom- panied by the change from the Fe(II)–O 2 to the Fe(III) complexes of the wild-type (black) and L99T mutant (red) proteins of Ec DosH. Experimental data (dotted lines) are fitted to the calculated lines with the auto-oxidation rate constants of 0.0063 and 0.049 min )1 , for the wild-type and L99T mutant, respectively. Table 2. Optical absorption spectral maxima (nm) of the Fe(II)–O 2 complexes and auto-oxidation rates (k ox ) of the wild-type and mutant Ec DosH proteins. Half-lives of the Fe(II)–O 2 complexes are also described in the right column. ND, no data. Soret (nm) b (nm) a (nm) k ox (min )1 ) t 1 ⁄ 2 (min) References Wild-type 417 541 579 0.0063 110 This study – – – 0.0058 120 [5] – – – 0.0053 130 [13] L99T 417 541 579 0.049 14 This study L99F a ND 0.37 1.9 This study L115T 417 541 579 0.065 11 This study L115F 417 541 579 0.33 2.1 This study M95A – – – 0.0013 530 [5] M95L – – – 0.0017 410 [5] M95H – – – 0.016 43 [5] D40A – – – 0.051 14 [13] D40N – – – 0.033 21 [13] a Fast O 2 dissociation (or fast auto-oxidation) may have hampered the determination of exact values because the O 2 binding rate was similar to that of the wild-type (Table 3). Heme electronic states of Leu mutants of Ec DOS N. Yokota et al. 1214 FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS with new PC and software, which allowed us to mon- itor broader time domains simultaneously and thus probably to detect both fast and slow phases. The reason why the kinetics of O 2 binding is composed of two phases is not currently clear. The crystal struc- ture of the Fe(II)–O 2 complex in Ec DosH forms a homodimer [2], and one O 2 molecule can bind to only one subunit of the dimer [10]. Therefore, it is possible that the first O 2 molecule binds quickly to one of the dimers, followed by binding of the second O 2 molecule to the other subunit of the dimer, result- ing in two phases. However, further study is needed to probe this possibility, as there is no obvious struc- tural origin for the differences in binding to the monomers. Another possibility is that once the O 2 molecule binds to the heme distal site and the distal Arg97 binds to the O 2 molecule [10], it may allosteri- cally influence the binding of the second O 2 molecule. (Table 3). It is not clear why a stable Fe(II)–O 2 complex was not observed for the L99F mutant (Table 2). The O 2 binding rate to the L99F mutant was similar to those of the wild-type and the other mutant proteins Table 3. Rates for O 2 association with the wild-type and mutant Ec DosH proteins as measured by the stopped-flow method. Rates determined by the stopped-flow method were dependent on the O 2 concentration. At least three experiments were conducted to obtain each rate constant. Experimental errors were less than 20%. ND, no data. k on (· 10 )3 lM )1 Æs )1 ) References Fast phase Slow phase Wild-type 81 8.3 This study 31 – [5] – 2.6 [1] L99T 49 6.8 This study L99F a 75 ND This study L115T 55 7.2 This study L115F b ND ND This study M95A > 1000 – [5] M95L > 1000 – [5] M95H > 1000 – [5] M95I > 1000 – [5] a The slow phase of the L99F mutant was independent of the O 2 concentration. b Measurement of the rate of O 2 binding was not feasible because of low heme binding affinity and instability of the protein. A C B D Fig. 5. (A) Changes in the optical spectra of the Fe(II)–O 2 complex formation of the wild-type protein after mixing solutions of protein (8 lM per heme) and O 2 (488 lM) in the stopped-flow spectrometer. (B) The spectral changes monitored at 429 nm accompanied by the O 2 associ- ation with the Fe(II) complex were composed of two phases. Experimental data (red dotted line) were fitted using a two-phase model (black solid line). Rates for the O 2 association for both the fast (C) and slow (D) phases were dependent on the O 2 concentration. N. Yokota et al. Heme electronic states of Leu mutants of Ec DOS FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS 1215 (Table 3). In addition, the rate of auto-oxidation for L99F was fast but comparable to that of the L115F mutant (Table 2). The high rate constant for the disso- ciation of O 2 from the L99F mutant compared with the other proteins along with the high rate of auto- oxidation explains why we did not monitor the stable Fe(II)–O 2 complex. CO binding kinetics We next examined the kinetics of CO binding for the mutant proteins of Ec DosH using laser flash photo- lysis (Fig. 6 and Table 4). The changes in the Soret region of the absorption spectra did not exhibit simple isosbestic points, but were apparently composed of two sets of spectral changes (Fig. 6A). Specifically, the spectral changes in the fast phase had isosbestic points around 414, 430, and 462 nm (red in Fig. 6B), whereas those in the slow phase had isosbestic points around 396, 425, and 450 nm (blue in Fig. 6C). We overlapped the spectral changes associated with the fast (isosbestic points at 430 nm) and slow (isosbestic point at 425 nm) reactions (Fig. 6B,C). The spectral changes associated with the fast phase occurred within a time scale of microseconds, whereas those of the slow phase were over a millisecond time scale. The pattern of CO association with Ec DosH monitored at 420 nm was also composed of fast and slow phases (Fig. 7A). The fast phases, which occurred of the order of microsec- onds (Fig. 7B), were independent of the CO concentra- tion, whereas the slow phases, which were of the order of milliseconds, were dependent on the CO concentra- tion (inset of Fig. 7B). The rate constants of the fast phase of the mutant proteins, except for the L115F mutant, were (5.7–6.3) · 10 4 s )1 , which is slightly higher than that of the wild-type protein (3.2 · 10 4 s )1 ). The rate constants of the CO-depend- ent slow phase were (29–44) · 10 )3 lm )1 Æs )1 , which is slightly higher than that of the wild-type protein (26 · 10 )3 lm )1 Æs )1 ) (Table 4). Therefore, the kinetics of CO binding was not markedly influenced by the mutation of Leu99 or Leu115. These findings are in contrast with the fact that mutations at the Met95 resi- due, the distal axial ligand in the Fe(II) complex, A B C Fig. 6. Transient spectra accompanying CO binding to the wild-type enzyme. (A) Difference absorption spectral changes of the wild-type protein (8 l M per heme) observed after flash photolysis. Spectra of the fast phase (red) obtained 0.6 ls, every few microseconds, and 200 ls after a flash are selected, and those of the slow phase (blue) obtained 0.2 ms, every few milliseconds and 80 ms after a flash are selected. Arrows designate spectral changes observed accompanied by the CO binding to the Fe(II) complex. Spectral changes were composed of fast (red) and slow (blue) components with different isosbestic points. (B) Difference spectral changes for the fast phase (of the order of microseconds) with isosbestic points at 414, 431, and 462 nm. (C) Difference spectral changes for the slow phase (of the order of milliseconds) with isosbestic points at 396, 425, and 450 nm. (B) and (C) were separately extracted from (A). Arrows in (B) and (C) designate the same as in (A). Heme electronic states of Leu mutants of Ec DOS N. Yokota et al. 1216 FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS markedly enhance the rate constants (Table 4) [5]. This was surprising because other physicochemical proper- ties such as the auto-oxidation rates and redox poten- tials (see next section) of the Leu99 and Leu115 mutant and wild-type proteins differed significantly. A possible explanation for these findings is that O 2 and CO have different modes of ligand binding but share a ligand access channel in Ec DosH. Intermediate species generated by flash photolysis As shown in Fig. S2 of Supplementary material, we observed spectral changes containing Soret peaks of two intermediate complexes [complexes b (435-nm spe- cies) and c (428-nm species)] and of the final Fe(II)– CO complex [complex a (423-nm species)] in the differ- ence spectra obtained from the absorbance before and after flash photolysis. The complex changes in the opti- cal absorption for the Fe(II)–CO complex after flash photolysis were similar to those previously reported for Ec DosH [16,18]. These previous studies proposed that the following species are obtained by flash photo- lysis. In the spectral changes for the fast phase, the peak around 438 nm is ascribed to the 5-coordinated intermediate complex (complex b in Fig. S2). The results suggest that the rate of binding of Met95 to the 5-coordinated Fe(II) complex (His77 as an axial lig- and; complex b in Fig. S2) is fast and occurs in the order of microseconds, whereas the rate of CO binding to the 6-coordinated Fe(II) complex (Met95 and His77 as axial ligands; complex c in Fig. S2) is slow and occurs in the order of milliseconds because CO must push the axial ligand out of the heme. For this slow CO binding process, CO is probably dissociated and moves to a position relatively far from the heme iron (complex b in Fig. S2). In addition to this slow CO binding process, the results indicate that the very fast CO binding should occur on a nanosecond timescale. For this very fast CO binding process, it is likely that CO does not move away but is located very close to the heme iron (complex d in Fig. S2). In this case, CO may not have sufficient time to move away after disso- ciation by flash photolysis, leading to the very fast recombination. Indeed, ultrafast ligand rebinding to Ec DosH has been reported [15,16]. A B Fig. 7. Optical spectral changes for the Fe(II)–CO complex of the wild-type protein (8 l M per heme) monitored at 422 nm after flash photolysis. Spectral changes were composed of two phases, fast (A) and slow (B) phases. Spectral changes for the slow phase were dependent on the CO concentration (inset), and the rate was evalu- ated as 26 · 10 )3 lM )1 Æs )1 . Table 4. Rates for CO association with the wild-type and mutant Ec DosH proteins as determined by the flash photolysis method. The fast phases were independent of the CO concentration, whereas the slow phases were dependent on the CO concentra- tion. Note that the fast phase is associated with rebinding of Met95 to the heme. At least three experiments were conducted to obtain each rate constant. Experimental errors were less than 20%. ND, no data. Fast phase k on (· 10 4 s )1 ) Slow phase k on (· 10 )3 lM )1 Æs )1 ) References Wild-type 3.2 26 This study – 7.8 [5] – 1.1 [1] L99T 6.3 29 This study L99F 5.7 44 This study L115T 6.3 41 This study L115F ND a ND a This study M95A – 9300 [5] M95L – 3400 [5] M95H – 6200 [5] M95I – 1100 [5] a Measurement of the rate of CO binding was not feasible because of low heme binding affinity and instability of the protein. N. Yokota et al. Heme electronic states of Leu mutants of Ec DOS FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS 1217 Redox potentials We obtained both the reductive and oxidative poten- tials for Ec DosH (Fig. 8 and Table 5). Both the reductive [23.1–34.6 mV versus the standard hydrogen electrode (SHE)] and oxidative potentials (17.7– 25.2 mV versus SHE) of the mutant proteins, except for the L115F mutant, were significantly lower than those of the wild-type protein (63.9 and 52.7 mV ver- sus SHE, respectively). It is surprising that the redox potential was decreased by the mutations at the Leu99 and Leu115 residues of Ec DosH because these resi- dues are distant from the heme plane and do not have direct contact with the heme iron. In our previous studies, mutations at Met95 and Asp40 of Ec DosH markedly changed the redox potentials (Table 4) [8,13]. Because Met95 is an axial ligand, Met95 muta- tions would be expected to give rise to marked changes in the redox potential. Also, Asp40 indirectly interacts with the proximal axial ligand, His77, of Ec DosH through ionic interactions via two water molecules. However, Leu99 and Leu115 have neither direct con- tact via ionic interaction nor covalent interaction. Leu99 should be located slightly farther from the heme iron than Leu115, but despite this, the changes in the redox potentials for the Leu99 mutants were larger than that for the L115T mutant. Intriguingly, the redox potentials for the Leu99 and Leu115 mutants of Ec DosH are in the opposite direc- tion from the Asp40 mutants, suggesting that the effects of mutations on the redox potential at the distal side differ from those at the heme proximal side. In this sense, it is reasonable that the Leu99 and Leu115 mutants decreased the redox potentials to extents sim- ilar to those observed for the Met95 mutations, although the effects of the Leu99 and Leu115 muta- tions were modest compared with those of Met95 mutations. It seems likely that potential data based on our data and that of others have accuracies of % 3–5 mV. Even taking the accuracy into consideration, redox poten- tials of Ec DosH proteins have an apparent hysteresis in the data, being different between the reductive and oxidative potentials (Table 5). The apparent hysteresis was small for the L99T mutant compared with the wild-type and other mutants. We do not know whether the hysteresis simply reflects differences in equilibration when data are recorded in reductive and oxidative directions. However, it is possible that it is due to the axial ligand switching between hydroxide anion and Met95 accompanied by the redox change as demon- A B Fig. 8. (A) Spectral changes of the L99F mutant accompanied by reduction by sodium dithionite. Arrows designate changes of the spectrum of the Fe(III) complex (black) to that of the Fe(II) complex (red). (B) Electrochemical reductive (black open circle) and oxidative (red filled circle) titrations of the L99F mutant of Ec DosH. Experi- mental data (dotted lines) are fitted to the calculated lines. Table 5. Redox potentials (mV versus SHE) of the wild-type and mutant Ec DosH proteins. We speculate that accuracies are % 3- 5 mV based on data of ours and others [2,8,13]. ND, no data. Reductive Oxidative References Wild-type 63.9 (n ¼ 0.88) 52.7 (n ¼ 0.98) This study 70 63 [2] 67 – [13] L99T 23.1 (n ¼ 0.92) 20.3 (n ¼ 1.01) This study L99F 24.2 (n ¼ 1.11) 17.7 (n ¼ 1.01) This study L115T 34.6 (n ¼ 0.93) 25.2 (n ¼ 1.08) This study L115F a ND ND This study M95L )1 – [8] M95A )26 – [8] M95H )122 – [8] D40A 95 – [13] D40N 114 – [13] a Measurements of redox potentials were not feasible because of low heme binding affinity and instability of the protein. Heme electronic states of Leu mutants of Ec DOS N. Yokota et al. 1218 FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS strated from the crystal structure of this protein [6]. In CooA, it was suggested that the hysteresis observed in the redox titrations reflects the axial ligand switching between Cys75 and His77 [25]. NO complexes Because the catalytic activity of Ec DOS may be strongly inhibited by NO binding to the heme iron [2], we examined the optical absorption spectra of the NO complex of the Ec DosH mutant proteins (Fig. 9 and Table 6). For the Fe(III)–NO complexes of the mutant proteins, we did not observe any significant differences between the wild-type and mutant proteins. Also, by adding sodium dithionite to the Fe(III) species, we confirmed that auto-reduction of the Fe(III)–NO com- plex to the Fe(II)–NO complex does not occur for the mutant proteins even under anaerobic conditions, which is consistent with the characteristics of the wild- type protein. Notably, however, the Soret absorption peak of the Fe(II)–NO complex of the L115F mutant was at 399 nm, which is different from those of the wild-type and other mutant proteins (421–423 nm). The Soret peak position of the Fe(II)–NO heme com- plex below or near 400 nm indicates a 5-coordinated heme–NO complex [14]. It is interesting to note that the Fe(II)–NO com- plex of the L115F mutant of Ec DosH is a 5-co- ordinated NO–heme complex. The heme-sensor enzyme, soluble guanylate cyclase, is activated by the formation of the 5-coordinated NO–heme complex [24]. A similar 5-coordinated NO–heme complex is formed for other heme-sensor enzymes and proteins, including CooA, cystathionine b-synthase, bacterial cytochrome c¢, and heme-regulated eukaryotic initi- ation factor 2a kinase [14,24 and references therein]. Many other heme proteins, including myoglobin, hemoglobin, peroxidases, and cytochromes P450, have 6-coordinated NO–heme complexes [21–24 and references therein]. The protein structures on the heme proximal side and ⁄ or the bond length between the heme iron and the proximal ligand may contrib- ute to the formation of the 5-coordinated NO–heme complex. We therefore speculate that the bond strength between the heme iron and His77 for the L115F mutant Ec DosH is weaker than those of the Ec DosH wild-type and other Leu99 and Leu115 mutant proteins, probably because of an indirect effect from the L115F mutations trans to the prox- imal side. Nitrite and nitrate anions have been impli- cated as being important in signal transduction by NO [17]. However, these anions did not change the optical absorption spectra of the wild-type or mutant proteins. A B Fig. 9. Optical absorption spectra of the Fe(III)–NO (black) and Fe(II)–NO (red) complexes of the wild-type (6.8 l M per heme) (A) and L115F (5.8 l M per heme) (B) mutant proteins of Ec DosH. The spectra of L99F and L115T are essentially the same as those of the wild-type. Table 6. Optical absorption maxima (nm) and millimolar absorption coefficients (m M )1 Æcm )1 ) of the NO complexes of the wild-type and mutant Ec DosH proteins. The millimolar absorption coefficients (shown in parentheses) were determined using the pyridine hemo- chromogen method. Fe(III)–NO Fe(II)–NO Soret baSoret ba Wild-type 420 (133) 532 568 421 (108) 540 578 L99T 420 (124) 534 568 421 (109) 543 579 L99F 421 (135) 533 568 423 (120) 547 580 L115T 420 (140) 533 567 421 (78) 544 576 L115F 421 (163) 534 568 399 (64) – – N. Yokota et al. Heme electronic states of Leu mutants of Ec DOS FEBS Journal 273 (2006) 1210–1223 ª 2006 The Authors Journal compilation ª 2006 FEBS 1219 [...]... equation: DAbsðtÞ ¼ AeÀkt þ BeÀkt ð1Þ where DAbs is the total intensity changes at a certain time, t, after mixing, A and B are initial intensities for each phase, and k is the Boltzman constant The rates of O2 recombination were plotted against the concentration of O2 Both phases were dependent on the O2 concentration EDTA had no significant effect on the ligand binding kinetics (data not shown) At least... structures of the L99T, L99F, and L115T mutants were similar to that of the wild-type enzyme We further found that the rates of auto-oxidation and the redox potentials were significantly changed by the mutations of these two residues It is interesting that these physicochemical values were changed by the mutations, because the mutated residues, Phe99 and Leu115, are distant from the heme iron and do not... directly interact with the O2 molecule by ionic interaction Surprisingly, however, the mutations at Leu99 and Leu115 did not significantly in uence the kinetics of O2 or CO binding Taken together, these results suggest that Leu99 and Leu115 play significant roles in determining the electronic states of the heme iron but are not important in the mode of O2 or CO binding and ⁄ or the structure of the ligand access... protein from Escherichia coli, is a direct oxygen sensor Biochemistry 39, 2685–2691 2 Sasakura Y, Hirata S, Sugiyama S, Suzuki S, Taguchi S, Watanabe M, Matsui T, Sagami I & Shimizu T (2002) Characterization of a direct oxygen sensor heme protein from Escherichia coli Effects of the heme redox states and mutations at the heme- binding site on catalysis and structure J Biol Chem 277, 23821–23827 FEBS Journal... Sagami I, Sasakura Y & Shimizu T (2003) Relationships between heme incorporation, tetramer formation, and catalysis of a heme- regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants J Biol Chem 278, 53105– 53111 5 Taguchi S, Matsui T, Igarashi J, Sasakura Y, Araki Y, Ito O, Sugiyama S, Sagami I & Shimizu T (2004) Binding of oxygen and carbon monoxide to a heme- regulated... Redox potential, autooxidation and catalytic control Eur J Biochem 271, 3937–3942 Igarashi J, Sato A, Kitagawa T, Yoshimura T, Yamauchi S, Sagami I & Shimizu T (2004) Activation of hemeregulated eukaryotic initiation factor 2a kinase by nitric oxide is induced by the formation of a five-coordinate NO -Heme complex: optical absorption, electron spin resonance, and resonance Raman spectral studies J Biol Chem... sulfate fractionation and dialysis, the protein was subjected to Ni2+ ⁄ nitrilotriacetate ⁄ agarose chromatography (Qiagen, Valencia, CA, USA) Final purification was by Sephadex G-75 column chromatography SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue R-250 revealed that the purified protein was more than 95% homogeneous (Supplementary Material, Fig S1) Optical absorption spectra Experiments... 2006 The Authors Journal compilation ª 2006 FEBS 1221 Heme electronic states of Leu mutants of Ec DOS N Yokota et al 3 Sato A, Sasakura Y, Sugiyama S, Sagami I, Shimizu T, Mizutani Y & Kitagawa T (2002) Stationary and timeresolved resonance Raman spectra of His77 and Met95 mutants of the isolated heme domain of a direct oxygen sensor from Escherichia coli J Biol Chem 277, 32650– 32658 4 Yoshimura T, Sagami... gtcgggagACCcagctggagaaaaaag-3¢, 5¢-gatgagtcgggagTTTcag ctggagaaaaaag-3¢, 5¢-ggacccgttttgcgACCtcgaaagtgagc-3¢, and 5¢-ggacccgttttgcgTTTtcgaaagtgagc-3¢ Preparation of Ec DosH The (His)6-tagged Ec DosH proteins (wild-type and L99T, L99F, L115T, and L115F mutants) were expressed in E coli BL21 (DE3) and purified as described previously [2,4] Heme synthesis was induced with 5-aminolevulinate (450 lm) After purification by 30–70% ammonium... USA) with the fundamental radiation of 1064 nm The monitoring light was produced using a 150 W xenon lamp (Hamamatsu Photonics, Hamamatsu, Japan) The peak power of the laser was 10 mJ with a pulse width of 6 ns (a repetition rate of 10 Hz) from a xenon lamp with the light intensity reduced by as much as 80% at 1 Delgado-Nixon VM, Gonzalez G & Gilles-Gonzalez MA (2000) Dos, a heme- binding PAS protein . Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme- regulated phosphodiesterase from Escherichia. DosH as a template and using the following respective 5¢-sense primers: 5¢-gatga gtcgggagACCcagctggagaaaaaag-3¢,5¢-gatgagtcgggagTTTcag ctggagaaaaaag-3¢,5¢-ggacccgttttgcgACCtcgaaagtgagc-3¢, and