Redox characteristics of a de novo quinone protein.
The electrochemistry of 2,6-dimethylbenzoquinone (DMBQ) has been characterized for three different systems: DMBQ freely solvated in aqueous buffer; DMBQ bound to a neutral, blocked cysteine (N-acetyl-L-cysteine methyl ester) and the resulting DMBQ-bCys compound solvated in aqueous buffer; and DMBQ bound to a small model protein denoted alpha(3)C. The goal of this study is to detect and characterize differences in the redox properties of the protein-ligated DMBQ relative to the solvated quinones. The alpha(3)C protein used here is a tryptophan-32 to cysteine-32 variant of the structurally defined alpha(3)W de novo protein (Dai et al. J. Am. Chem. Soc. 2002, 124, 10952-10953). The properties of alpha(3)C were recently described (Hay et al. Biochemistry 2005, 44, 11891-11902). DMBQ was covalently bound to bCys and alpha(3)C through a sulfur substitution reaction with the cysteine thiol. In contrast to the solvated DMBQ and DMBQ-bCys compounds, diffusion controlled electrochemistry of DMBQ-alpha(3)C showed well-behaved and fully reversible n = 2 oxidation/reduction with a peak separation of approximately 30 mV between pH 5 and 9. DMBQ-alpha(3)C could also be immobilized on a gold electrode modified with a self-assembled monolayer of 3-mercaptopropionoic acid, allowing the measurement, by cyclic voltammetry, of an apparent rate of electron transfer of 22 s(-1). The (cysteine) sulfur substitution significantly lowers one of the hydroquinone pKA's from 10.4 in DMBQ to 6.8 in DMBQ-bCys. This pKA is slightly elevated in DMBQ-alpha(3)C to 7.0 and the E1/2 at pH 7.0 is raised by 110 mV from +190 mV in DMBQ-bCys to +297 mV in DMBQ-alpha(3)C.
Hay
Sam
S
Department of Biochemistry & Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Westerlund
Kristina
K
Tommos
Cecilia
C
D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000737:chemistry*; D002021:Buffers; D003545:Cysteine / Q000737:chemistry; D004563:Electrochemistry; D004566:Electrodes; D004579:Electron Transport; D006046:Gold; D006873:Hydroquinones / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*
D016227:Benzoquinones; D002021:Buffers; D006873:Hydroquinones; D011506:Proteins; C505583:alpha3C protein; D006046:Gold; C035577:2,6-dimethyl-1,4-benzoquinone; D003545:Cysteine; C031927:hydroquinone