title: Tolerance of the Rieske-type [2Fe-2S] cluster in recombinant ferredoxin BphA3 from Pseudomonas sp. KKS102 to histidine ligand mutations.
abstract: BphA3 from Pseudomonas sp. KKS102 is a Rieske-type [2Fe-2S] ferredoxin that transfers electrons from an NADH-dependent oxidoreductase, BphA4, to a biphenyl dioxygenase complex. A high-level expression and purification system for the recombinant BphA3 in Escherichia coli was constructed. Two histidine ligands of the Rieske-type cluster in BphA3, were each replaced with serine, cysteine, asparagine and tyrosine. The single mutants, in which either His44 or His65 was replaced with a cysteine residue (CH and HC mutants respectively), and the double mutant, in which both histidine residues were replaced with cysteine residue (CC mutant), accumulated to high levels in the E. coli cells, while the other single mutants did not. The purified WT (wild-type) protein showed characteristic near-UV and visible absorption and CD spectra of Rieske-type clusters. The X-ray absorption spectra were suggestive of the existence of [2Fe-2S] clusters, with one histidine and three cysteine ligands in the CH and HC mutants, and an [2Fe-2S] cluster with four cysteine ligands in the CC mutant. The BphA4-dependent cytochrome c reductase activities of the mutants were less than 0.3% of that of the WT protein. The redox potential of the WT protein determined by cyclic voltammetry was -180+/-5 mV compared with the standard hydrogen electrode, and that of the CH mutant was approx. 175 mV lower. The changes in the near-UV and visible absorption spectra of the mutants showed that the reduced iron-sulphur clusters in the mutants were unstable. His44 and His65 in BphA3 can be replaced with cysteine residues, but are required for the stabilization of the reduced form of the cluster.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kamigori, Hyogo 678-1297, Japan. s-kimurar@sci.u-hyogo.ac.jp
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Kimura
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Toshiya
authors.initials: T
authors.lastname: Senda
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.forename: Masao
authors.initials: M
authors.lastname: Fukuda
mesh_terms: D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011549:Pseudomonas / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D006639:Histidine; D014450:Electron Transport Complex III
references.citation: J Biol Chem. 1985 Mar 25;260(6):3251-4
references.pmid: 2982852
references.citation: Biochim Biophys Acta. 1989 Jul 13;975(2):293-8
references.pmid: 2545269
references.citation: J Biol Chem. 1967 Feb 10;242(3):485-91
references.pmid: 4381523
references.citation: Biochemistry. 2000 Jun 27;39(25):7436-44
references.pmid: 10858292
references.citation: J Bacteriol. 1994 Jul;176(14):4269-76
references.pmid: 8021212
references.citation: Biokhimiia. 1995 Aug;60(8):1221-30
references.pmid: 7578577
references.citation: Biochemistry. 1999 Nov 30;38(48):15764-73
references.pmid: 10625442
references.citation: J Biol Chem. 2004 Mar 26;279(13):12519-28
references.pmid: 14726526
references.citation: Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):750-4
references.pmid: 1992467
references.citation: J Biol Chem. 1998 Apr 10;273(15):9085-93
references.pmid: 9535897
references.citation: Biochem Biophys Res Commun. 1994 Jul 29;202(2):850-6
references.pmid: 8048958
references.citation: Biochemistry. 1967 Jun;6(6):1658-64
references.pmid: 4292055
references.citation: J Mol Biol. 2000 Dec 1;304(3):397-410
references.pmid: 11090282
references.citation: Biochemistry. 1994 Mar 22;33(11):3155-64
references.pmid: 8136349
references.citation: J Biol Chem. 1984 Jan 10;259(1):124-33
references.pmid: 6323399
references.citation: Biochemistry. 1992 Apr 7;31(13):3342-51
references.pmid: 1313292
references.citation: Structure. 2000 Dec 15;8(12):1267-78
references.pmid: 11188691
references.citation: Biochemistry. 2001 Jan 9;40(1):84-92
references.pmid: 11141059
references.citation: J Biochem. 2003 Sep;134(3):403-13
references.pmid: 14561726
references.citation: J Mol Biol. 1985 Oct 20;185(4):713-20
references.pmid: 2414455
references.citation: J Bacteriol. 1994 Mar;176(6):1689-94
references.pmid: 8132464
references.citation: J Mol Biol. 1999 May 14;288(4):521-38
references.pmid: 10329160
references.citation: Biochemistry. 1995 Dec 26;34(51):16533-42
references.pmid: 8527426
references.citation: Biochemistry. 1996 Jun 11;35(23):7546-52
references.pmid: 8652534
references.citation: Biochemistry. 1989 May 30;28(11):4861-71
references.pmid: 2765515
references.citation: Biochemistry. 1998 Mar 17;37(11):3965-73
references.pmid: 9521718
references.citation: J Bacteriol. 1989 May;171(5):2740-7
references.pmid: 2540155
references.citation: Eur J Biochem. 1998 Jul 1;255(1):100-6
references.pmid: 9692907
references.citation: Biochim Biophys Acta. 1999 Mar 19;1430(2):290-301
references.pmid: 10082957
references.citation: Protein Sci. 2002 Dec;11(12):2969-73
references.pmid: 12441394
references.citation: Science. 1997 Aug 1;277(5326):653-9
references.pmid: 9235882
references.citation: Anal Biochem. 1987 Nov 1;166(2):368-79
references.pmid: 2449095
references.citation: Annu Rev Microbiol. 1992;46:277-305
references.pmid: 1444257