Redox titration using fluorescence measurements of photosystem II (PSII) has long shown that impairment of the water-oxidizing Mn4CaO5 cluster upshifts the redox potential (Em) of the primary quinone electron acceptor QA by more than 100 mV, which has been proposed as a photoprotection mechanism of PSII. However, the molecular mechanism of this long-distance interaction between the Mn4CaO5 cluster and QA in PSII remains unresolved. In this study, we reinvestigated the effect of depletion of the Mn4CaO5 cluster on Em(QA−/QA) using Fourier transform infrared (FTIR) spectroelectrochemistry, which can directly monitor the redox state of QA at an intended potential. Light-induced FTIR difference measurements at a series of electrode potentials for intact and Mn-depleted PSII preparations from spinach and Thermosynechococcus elongatus showed that depletion of the Mn4CaO5 cluster hardly affected the Em(QA−/QA) values. In contrast, fluorescence spectroelectrochemical measurement using the same PSII sample, electrochemical cell, and redox mediators reproduced a large upshift of apparent Em upon Mn depletion, whereas a smaller shift was observed when weaker visible light was used for fluorescence excitation. Thus, the possibility was suggested that the measuring light for fluorescence disturbed the titration curve in Mn-depleted PSII, in contrast to no interference of infrared light with the PSII reactions in FTIR measurements. From these results, it was concluded that the Mn4CaO5 cluster does not directly regulate Em(QA−/QA) to control the redox reactions on the electron acceptor side of PSII.