Structural and kinetic studies of imidazole binding to two members of the cytochrome c (6) family reveal an important role for a conserved heme pocket residue.
The amino acid at position 51 in the cytochrome c(6) family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c(6) Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe-S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k (obs) for imidazole binding to Arabidopsis thaliana cytochrome c(6A), which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c(6), was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s(-1)). For the cytochrome c(6) Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c(6A) was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c(6) family is additionally responsible for tuning the stability of the heme iron-Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding.
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK.
Badri S
BS
Rajagopal
Michael T
MT
Wilson
Derek S
DS
Bendall
Christopher J
CJ
Howe
Jonathan A R
JA
Worrall
D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000201:enzymology; D045365:Cytochromes c6 / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure
D045365:Cytochromes c6; D007093:Imidazoles; D006418:Heme; C029899:imidazole
J Biol Inorg Chem. 2008 Jun;13(5):837-45
18392863
Eur J Biochem. 2001 Aug;268(16):4468-76
11502207
Biochemistry. 1999 Apr 6;38(14):4480-92
10194370
Biochemistry. 1986 Apr 8;25(7):1675-81
3011070
Biochemistry. 2001 Aug 7;40(31):9215-25
11478889
Structure. 1995 Nov 15;3(11):1159-69
8591027
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8312-7
10880578
Biochem J. 1979 Feb 1;177(2):641-8
35158
FEBS J. 2009 Aug;276(16):4426-36
19678839
Acta Crystallogr D Biol Crystallogr. 2005 Apr;61(Pt 4):458-64
15805601
Science. 1995 Jul 14;269(5221):192-7
7618079
Nature. 2003 Jul 3;424(6944):33-4
12840749
J Synchrotron Radiat. 2008 Sep;15(Pt 5):433-9
18728313
Biochemistry. 2002 Dec 17;41(50):14689-99
12475218
Arch Biochem Biophys. 1999 Nov 15;371(2):142-8
10545200
FEBS Lett. 2006 Jun 26;580(15):3763-8
16777100
FEBS Lett. 1989 Mar 27;246(1-2):105-8
2540029
Nat Struct Biol. 1997 Jan;4(1):51-6
8989324
Biochemistry. 2005 Apr 12;44(14):5488-94
15807542
J Am Chem Soc. 2007 Aug 1;129(30):9468-75
17625855
Eur J Biochem. 1978 Jun 1;87(1):9-19
208838
J Mol Biol. 1995 Jul 28;250(5):627-47
7623381
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
J Mol Biol. 2000 May 19;298(5):955-69
10801361
Methods Enzymol. 1997;276:307-26
27754618
Proteins. 2008 Nov 15;73(3):690-704
18491383
Proc Natl Acad Sci U S A. 2005 Aug 23;102(34):12047-52
16103362
J Mol Biol. 2003 Aug 1;331(1):37-43
12875834
Biochemistry. 1995 Sep 12;34(36):11385-98
7547866
Mol Cell Biol. 1986 Feb;6(2):462-9
3023849
J Am Chem Soc. 2002 Oct 2;124(39):11574-5
12296704
Biochemistry. 2004 Jun 22;43(24):7717-24
15196014
J Biol Inorg Chem. 2001 Jun;6(5-6):628-37
11472026
Acta Crystallogr D Biol Crystallogr. 1997 Jul 1;53(Pt 4):448-55
15299911
J Biol Chem. 1972 Nov 10;247(21):6932-6
4343163
J Am Chem Soc. 2003 Mar 12;125(10):2913-22
12617658
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
J Mol Biol. 1998 Feb 6;275(5):719-24
9480763
Biochim Biophys Acta. 2004 Jul 9;1657(2-3):115-20
15238268
Acta Crystallogr D Biol Crystallogr. 2000 Dec;56(Pt 12):1577-82
11092924
Acta Crystallogr D Biol Crystallogr. 1994 Jul 1;50(Pt 4):596-602
15299423
Trends Biochem Sci. 2001 Sep;26(9):521-3
11551776
Arch Biochem Biophys. 2002 Sep 15;405(2):154-62
12220527
J Biol Chem. 1965 Sep;240(9):3660-3
5835945
J Biol Chem. 1998 Oct 2;273(40):25647-53
9748230
Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4246-9
9113974
Biochemistry. 1969 Jan;8(1):149-53
5777317
J Synchrotron Radiat. 2007 Jan;14(Pt 1):11-23
17211068
Chem Rev. 2004 Feb;104(2):589-615
14871136
Biochemistry. 2008 May 20;47(20):5515-22
18439023
Proc Natl Acad Sci U S A. 2005 Mar 29;102(13):4741-6
15774579
J Mol Biol. 1999 Jul 30;290(5):1019-30
10438600
J Am Chem Soc. 2003 Nov 12;125(45):13650-1
14599189
Biochemistry. 1994 Jun 7;33(22):6925-35
8204626
J Mol Biol. 2006 Jul 28;360(5):968-77
16815443