Redox Properties and Coordination Structure of the Heme in the CO-sensing Transcriptional Activator CooA

• DOI: 10.1074/jbc.m003972200
• Publication Date: 2002-07-26T15:09:37Z
• Authors: Hiroshi Nakajima; Yumiko Honma; Toshifumi Tawara; Toshiyuki Kato; Sam-Yong Park; Hideyuki Miyatake; Yoshitsugu Shiro; Shigetoshi Aono
• Keywords: Hemeprotein

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as CO sensor. Cys75 and His77 are assigned to the fifth ligand of ferric ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis EXAFS analyses determine coordination structure heme in CooA. Pro2 is thought be sixth hemes CooA, which consistent with crystal (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876–880). exhibited anomalous redox chemistry, i.e. hysteresis was observed electrochemical titrations reduction oxidation midpoint potentials were −320 mV −260 mV, redox-controlled exchange between cause difference potentials. soluble guanylate cyclase extended x-ray absorption fine electron paramagnetic resonance normal hydrogen electrode Hemeproteins most popular metalloproteins exhibiting wide variety functions such oxygen storage/transport, transfer, reactions various substrates (1Lippard S.J. Berg J.M. Principles Bioinorganic Chemistry. University Science Books, Mill Valley, CA1994Google Scholar, 2Yamanaka Biochemistry Bacterial Cytochromes. Japan Scientific Societies Press/Springer-Verlag, Tokyo1992Google Scholar). addition these traditional proteins, new class proteins termed heme-based sensor have been reported recently acts an effector molecule (3Rodgers K.R. Curr. Opin. Chem. 1999; 3: 158-167Crossref PubMed Scopus (201) Google There five known, (sGC)1 (4Craven P.A. 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On hand, ferric, ferrous, CO-bound forms (26Aono Nakajima Saito Okada 228: 752-756Crossref (109) 28Uchida Ishikawa Ishimori Morishima I. Ohkubo Aono 273: 19988-19992Abstract 29Aono Matsuo 25757-25764Abstract (111) 34Shelver Kerby R.L. He Roberts G.P. 1997; 94: 11216-11220Crossref (192) 35Shelver Thorsteinsson M.V. Chung S.-Y. Reynolds M.F. Parks R.B. Burstyn 38: 2669-2678Crossref (100) 36Vogel K.M. Spiro Shelver 2679-2687Crossref (72) 37Reynolds Park 120: 9080-9081Crossref Therefore, must replace one axial ligands when its (28Uchida upon binding functionally relevant for described below. physiological regulating activity CooA;i.e. only form can bound target DNA active (27Aono Shimono Takasaki 240: 783-786Crossref (26) Scholar,31Shelver Bacteriol. 177: 2157-2163Crossref 32He 271: 120-123Abstract (86) 33Fox J.D. Ludden P.W. 178: 6200-6208Crossref Replacement triggers activation release causes conformational changes around subsequently whole Scholar,35Shelver induced by interchange principal part CO. Because plays central role elucidation required understand mechanisms Mutagenesis studies, EPR, Raman, uv/vis spectroscopies revealed low-spin respectively 38Dhawan I.K. Johnson 12805-12813Crossref (49) results suggest possibility takes place during change (29Aono Recently, Lanzilotta et al. (39Lanzilotta W.N. Schuller D.J. Poulos T.L. 7: 876-880Crossref Scholar) shows N-terminal subunit provides homodimer has reported, remains elucidated. also controversial. We proposed whereas Vogel (34Shelver Dhawan (38Dhawan pointed displaces report herein analyses. examined properties means titrations. Site-directed using Quick-change site-directed kit (Stratagene). expression purification recombinant previously A Sephacryl S-100 (Amersham Pharmacia Biotech) gel filtration column equilibrated 50 mm Tris-HCl buffer, pH 8.0 containing 100 NaCl used final step purification. purified concentrated about 30 μm 2 ultrafiltration YM-10 membrane (Amicon, Inc.) titration measurements, gene encoding CooAΔN5 synthesized polymerase chain reaction pKK3CO5 template following primers: primer 1, 5′-AGGAGACTCGTATGAACATCGCCAATGTCCTGTTG-3′ 2, 5′-TCATTAATCGTCGTCGTCGTCGCGGTC-3′. PCR product cooAΔN5 cloned into pCR2.1 vector TA cloning (Invitrogen) construct pCR-COΔN5. An CooAΔN5, pKK-COΔN5, constructed inserting EcoRI fragment gene, cut pCR-COΔN5, pKK223-3 Biotech). measured experiments made cell equipped quartz optical (40 (height) × 10 (width) 1 (thickness)). path length mm. working gold mesh 9 0.7 mm) immersed cell. platinum wire Ag2+/AgCl (3 m KCl) electrodes (RE-1B, BSA) auxiliary reference electrodes, potential controlled potentiostat (HA-301, Hokuto Denko Co.). electronic spectra on U-3300 Hitachi spectrophotometer. For titrations, mediator dyes (2 each) added sample solution (the values E1/2 (versus NHE) shown parentheses): phenazine methosulfate (+80 Ref. 40Zahn Arciero D.M. Hooper A.B. Dispirito A.A. Eur. 684-691Crossref (35) gallocyanine (+20 indigo trisulfonate (−80 2-hydroxy-1, 4-naphthoquinone (−120 41Baymann F. Moss D.A. Mäntele Anal. 199: 269-274Crossref (50) anthraquinone 2-sulfonate (−230 benzyl viologen (−350 methyl (−440 42Okura Kaji N. Kita Yamada Inorg. 1985; 24: 451-453Crossref (40) N,N′-dimethyl-2,2′-bipyridinium hexafluorophosphate (−720 repeatedly degassed flushed argon prior measurement then ∼2.5 ml transferred cell, sealed rubber septum, gas-tight syringe under atmosphere. kept thermoelectric holder spectrophotometer 15 °C titration. followed recording absorbance regions Soret Q bands. iron K-edge liquid nitrogen temperature monochromatized synchrotron radiation BL 12C Photon Factory National Laboratory High Energy Physics (Tsukuba, Japan). fluorescence excitation bent cylinder type focusing mirror silicon double-crystal monochromator. placed angle 45 degrees against incident beam, fluorescent intensity perpendicular beam solid 19-element detector (Canberra Industries, analysis data performed program XFIT (43Ellis Freeman H.C. Synchrotron Rad. 2: 190-195Crossref (44Rich A.M. Armstrong R.S. Ellis Lay 37: 5743-5753Crossref (39) 45Rich 10827-10836Crossref (75) 46Rich Tennant L Wright P.E. 16491-16499Crossref (22) k-windows figures. multiple scattering outershell atoms porphyrin ring molecules taken account. constrained refinement, number parameters reduced treating set unit (47Obayashi Shoun 119: 7807-7816Crossref spectroscopic studies 30Aono Coord. Rev. 190–192: 267-282Crossref (36) To date, not His, Met, Cys, Lys candidates except identify unknown all Arg, Asp, Glu, Asn, Gln, Ser, Thr, Tyr residues heme-binding domain present work. chains could potentially coordinate iron, located region Met1to Met131 mutant work summarized Table individual partially Q-Sepharose their forms. All mutants thus prepared almost same did wild-type (data shown), indicating no candidate found mutagenesis. 2Among work, N6A D72A showed slightly blue-shifted peak 420 nm state, identical those However, Asn6 Asp72 replaced resulting any amino acid residues, His77, do CooA.Table IMutant workTarget residuesMutant proteinsArgR4A, R21A, R24A, R51A, R53A, R61A, R87A, R91A, R96A, R118A, R125AAspD15A, D40A, D49A, D72A, D94A, D129AGluE17A, E38A, E41A, E59A, E60A, E62A, E83A, E86A, E89A, E99A, E128AAsnN6A, N9A, N42AGlnQ100A, Q103ASerS13A, S25A, S32A, S64A, S70A, S78A, S107A, S122AThrT18A, T36A, T69A, T85A, T88A, T97A, T104A, T121A, T126ATyrY55F, Y67FThe numbers stand positions mutation introduced. Ala Tyr. Y55F Y67F mutants, Phe. Open table tab elucidate whether group amino-terminal residue coordinated truncated (CooAΔN5), four deleted measured. sequences deduced sequences, are1MPPRFNIANV 1MNIANV, underlined sequence CooAΔN5. 422, 424, 422 forms, respectively, Fig. 1. spectral features corresponding ones though broad shoulder 399 compared wild-type. CT-band 638 spectrum high-spin exists some extent 13.2 0.29 units/mg protein presence absence CO, comparable CooA; 15.7 0.23 CO-dependent deletion perturb result (48Thorsteinsson Conrad Youn Staples C.R. Serate 275: 39332-39338Abstract (38) above suggested first terminus responsible conclusion now known incorrect. During course reviewing process paper, atom Though indicate compensation maintain order it what analyze