Dry micrometer-thick crystalline photosystem I (PSI) has been shown to generate unprecedented large photovoltage under illumination. We use variable-temperature Kelvin probe force microscopy to show that deep acceptor centers are responsible for this anomalous photovoltage. We assumed that these centers are located close to the positively charged F(B)(2+) clusters, forming a coupled center that effectively captures the photoexcited electron into a deep state. We extract the main inherent parameters of the deep centers, which are extremely important in the potential use of photosynthetic proteins in various optoelectronic devices.