Cytochrome c (cyt c) was adsorbed on N-acetylcysteine (NAC)-modified gold electrodes via electrostatic interaction. The cyt c layer exhibited reversible and stable electrochemical redox transformation in 0.01 M phosphate butter, pH 7.4, where the heterogeneous electron transfer (ET) constant k＇(het) was measured by three techniques: cyclic voltammetry at high sweep rates (CV), electrochemical impedance (EI), and electroreflectance (ER) spectroscopy. In addition, k＇(het) was also determined from combining sets of simultaneous electrochemical impedance (EI) and electroreflectance (ER) measurements in a new impedance model in which a constant-phase element was used. The negligible shift(-0.023 mV) in the formal potential from the solution value, and the dose agreement of the measured distribution around the CV peaks (full-width voltage at half-peak-height, E-fwhh = 97 mV) with the theoretical value of 90.6 mV, suggested that the immobilized cyt c is retained at the electrode in the native state. Apparent k＇(het) values, as determined by each method separately, were as follows: (k＇(CV) = 920 +/- 280 s(-1) by CV, k＇(EI) = 660 +/- 200 s(-1) by EI, and k＇(ER) = 2100 +/- 300 s(-1) by ER as interpreted using previously published methods.(14,15,30) In the combined EI/ER measurements, k＇ER was found to increase linearly with the frequency of the sc modulating current and spanned a range from about 400 to 800 s(-1), which is close to the interprotein electron-transfer rate constant (800 s(-1)) measured between cyt c and one of its natural redox partners, cytochrome c peroxidase.(31) It is concluded that further attempts to reconcile these discrepancies in k＇(het) determinations will require more detailed descriptions of the interfacial elements in the impedance models.