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-acetylL-cysteine methyl ester) and the resulting DMBQ-bCys compound solvated in aqueous buffer; and DMBQ bound to a small model protein denoted alpha C-3. 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 C-3 protein used here is a tryptophan-32 to cysteine- 32 variant of the structurally defined alpha W-3 de novo protein (Dai et al. J. Am. Chem. Soc. 2002, 124, 10952-10953). The properties of alpha C-3 were recently described (Hay et al. Biochemistry 2005, 44, 11891-11902). DMBQ was covalently bound to bCys and alpha C-3 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 C-3 showed well- behaved and fully reversible n = 2 oxidation/reduction with a peak separation of similar to 30 mV between pH 5 and 9. DMBQ-alpha C-3 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 pK(A)'s from 10.4 in DMBQ to 6.8 in DMBQ-bCys. This pK(A) is slightly elevated in DMBQ-alpha C-3 to 7.0 and the E-1/2 at pH 7.0 is raised by 110 mV from + 190 mV in DMBQ-bCys to + 297 mV in DMBQ-alpha C-3.