Tolerance of the Rieske-type [2Fe-2S] cluster in recombinant ferredoxin BphA3 from Pseudomonas sp. KKS102 to histidine ligand mutations.
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.
Kimura
Shigenobu
S
Graduate School of Life Science, University of Hyogo, Kamigori, Hyogo 678-1297, Japan. s-kimurar@sci.u-hyogo.ac.jp
Kikuchi
Akihiro
A
Senda
Toshiya
T
Shiro
Yoshitsugu
Y
Fukuda
Masao
M
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
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D006639:Histidine; D014450:Electron Transport Complex III
J Biol Chem. 1985 Mar 25;260(6):3251-4
2982852
Biochim Biophys Acta. 1989 Jul 13;975(2):293-8
2545269
J Biol Chem. 1967 Feb 10;242(3):485-91
4381523
Biochemistry. 2000 Jun 27;39(25):7436-44
10858292
J Bacteriol. 1994 Jul;176(14):4269-76
8021212
Biokhimiia. 1995 Aug;60(8):1221-30
7578577
Biochemistry. 1999 Nov 30;38(48):15764-73
10625442
J Biol Chem. 2004 Mar 26;279(13):12519-28
14726526
Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):750-4
1992467
J Biol Chem. 1998 Apr 10;273(15):9085-93
9535897
Biochem Biophys Res Commun. 1994 Jul 29;202(2):850-6
8048958
Biochemistry. 1967 Jun;6(6):1658-64
4292055
J Mol Biol. 2000 Dec 1;304(3):397-410
11090282
Biochemistry. 1994 Mar 22;33(11):3155-64
8136349
J Biol Chem. 1984 Jan 10;259(1):124-33
6323399
Biochemistry. 1992 Apr 7;31(13):3342-51
1313292
Structure. 2000 Dec 15;8(12):1267-78
11188691
Biochemistry. 2001 Jan 9;40(1):84-92
11141059
J Biochem. 2003 Sep;134(3):403-13
14561726
J Mol Biol. 1985 Oct 20;185(4):713-20
2414455
J Bacteriol. 1994 Mar;176(6):1689-94
8132464
J Mol Biol. 1999 May 14;288(4):521-38
10329160
Biochemistry. 1995 Dec 26;34(51):16533-42
8527426
Biochemistry. 1996 Jun 11;35(23):7546-52
8652534
Biochemistry. 1989 May 30;28(11):4861-71
2765515
Biochemistry. 1998 Mar 17;37(11):3965-73
9521718
J Bacteriol. 1989 May;171(5):2740-7
2540155
Eur J Biochem. 1998 Jul 1;255(1):100-6
9692907
Biochim Biophys Acta. 1999 Mar 19;1430(2):290-301
10082957
Protein Sci. 2002 Dec;11(12):2969-73
12441394
Science. 1997 Aug 1;277(5326):653-9
9235882
Anal Biochem. 1987 Nov 1;166(2):368-79
2449095
Annu Rev Microbiol. 1992;46:277-305
1444257