title: Multiplexed electrochemistry of DNA-bound metalloproteins.
abstract: Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of endonuclease III (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Catrina G
authors.initials: CG
authors.lastname: Pheeney
authors.forename: Anna R
authors.initials: AR
authors.lastname: Arnold
authors.forename: Michael A
authors.initials: MA
authors.lastname: Grodick
authors.forename: Jacqueline K
authors.initials: JK
authors.lastname: Barton
mesh_terms: D004247:DNA / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055664:Electrochemical Techniques; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017354:Point Mutation
chemical_list: D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D004247:DNA; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli
references.citation: Protein Sci. 1999 Apr;8(4):841-53
references.pmid: 10211830
references.citation: Nat Struct Biol. 2002 Mar;9(3):188-92
references.pmid: 11875515
references.citation: Proc Natl Acad Sci U S A. 2012 Feb 7;109(6):1856-61
references.pmid: 22308447
references.citation: Angew Chem Int Ed Engl. 1999 Apr 1;38(7):941-945
references.pmid: 29711858
references.citation: Angew Chem Int Ed Engl. 2011 Apr 26;50(18):4137-41
references.pmid: 21472916
references.citation: J Am Chem Soc. 2004 Nov 24;126(46):15010-1
references.pmid: 15547981
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5002-6
references.pmid: 11296248
references.citation: Biochemistry. 1989 May 16;28(10):4450-5
references.pmid: 2548577
references.citation: J Am Chem Soc. 2011 Oct 19;133(41):16378-81
references.pmid: 21939244
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 8;102(10):3558-63
references.pmid: 15738426
references.citation: Nat Chem. 2011 Mar;3(3):228-33
references.pmid: 21336329
references.citation: Science. 1993 Nov 12;262(5136):1025-9
references.pmid: 7802858
references.citation: Nat Chem. 2012 Jun 03;4(8):642-8
references.pmid: 22824896
references.citation: Anal Chem. 2012 Jan 17;84(2):1098-103
references.pmid: 22145706
references.citation: Science. 2009 Jan 16;323(5912):352
references.pmid: 19150837
references.citation: Angew Chem Int Ed Engl. 2012 May 14;51(20):4896-900
references.pmid: 22488842
references.citation: Chem Rev. 2010 Mar 10;110(3):1642-62
references.pmid: 20214403
references.citation: J Am Chem Soc. 2006 Sep 20;128(37):12082-3
references.pmid: 16967954
references.citation: Bioconjug Chem. 2005 Mar-Apr;16(2):312-21
references.pmid: 15769084
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13164-8
references.pmid: 19651620
references.citation: Proc Natl Acad Sci U S A. 2009 Sep 8;106(36):15237-42
references.pmid: 19720997
references.citation: Nat Biotechnol. 2000 Oct;18(10):1096-100
references.pmid: 11017050
references.citation: Trends Biotechnol. 2011 Jan;29(1):1-5
references.pmid: 21106266
references.citation: Biochemistry. 2011 Jul 12;50(27):6133-45
references.pmid: 21651304
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 17;109(29):11528-33
references.pmid: 22733728
references.citation: Nat Struct Biol. 2002 Jan;9(1):48-52
references.pmid: 11740504
references.citation: J Am Chem Soc. 2012 Feb 22;134(7):3346-8
references.pmid: 22313286
references.citation: ACS Nano. 2009 Oct 27;3(10):3207-13
references.pmid: 19736919
references.citation: J Am Chem Soc. 2004 Oct 20;126(41):13168-9
references.pmid: 15479039
references.citation: J Am Chem Soc. 2009 Apr 1;131(12):4262-6
references.pmid: 19271708
references.citation: Science. 2005 Nov 25;310(5752):1311-3
references.pmid: 16311331
references.citation: EMBO J. 2003 Jul 1;22(13):3461-71
references.pmid: 12840008
references.citation: Biochemistry. 2005 Jun 14;44(23):8397-407
references.pmid: 15938629
references.citation: J Am Chem Soc. 2010 Mar 3;132(8):2769-74
references.pmid: 20131780
references.citation: Cell Mol Life Sci. 2009 Apr;66(7):1209-22
references.pmid: 19099189
references.citation: Curr Opin Struct Biol. 2012 Feb;22(1):94-100
references.pmid: 22169085
references.citation: Anal Chem. 2012 Jan 3;84(1):21-5
references.pmid: 22142422
references.citation: Langmuir. 2012 May 1;28(17):7063-70
references.pmid: 22512327
references.citation: FEBS Lett. 2012 Mar 9;586(5):526-35
references.pmid: 22024483
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 3;107(31):13684-9
references.pmid: 20643958
references.citation: Anal Chem. 2003 Nov 15;75(22):6327-33
references.pmid: 14616017
references.citation: EMBO J. 1995 Aug 15;14(16):4108-20
references.pmid: 7664751
references.citation: Nat Biotechnol. 2005 Oct;23(10):1294-301
references.pmid: 16170313
references.citation: Nucleic Acids Res. 2012 May;40(10):4247-60
references.pmid: 22287629