Photosystem II (PSII) is a large membrane bound molecular machine that catalyzes light-driven oxygen evolution from water. PSII constantly undergoes assembly and disassembly because of the unavoidable damage that results from its normal photochemistry. Thus, under physiological conditions, in addition to the active PSII complexes, there are always PSII subpopulations incompetent of oxygen evolution, but are in the process of undergoing elaborate biogenesis and repair. These transient complexes are difficult to characterize because of their low abundance, structural heterogeneity, and thermodynamic instability. In this study, we show that a genetically tagged Psb27 protein allows for the biochemical purification of two monomeric PSII assembly intermediates, one with an unprocessed form of D1 (His27ΔctpAPSII) and a second one with a mature form of D1 (His27PSII). Both forms were capable of light-induced charge separation, but unable to photooxidize water, largely because of the absence of a functional tetramanganese cluster. Unexpectedly, there was a significant amount of the extrinsic lumenal PsbO protein in the His27PSII, but not in the His27ΔctpAPSII complex. In contrast, two other lumenal proteins, PsbU and PsbV, were absent in both of these PSII intermediate complexes. Additionally, the only cytoplasmic extrinsic protein, Psb28 was detected in His27PSII complex. Based on these data, we have presented a refined model of PSII biogenesis, illustrating an important role of Psb27 as a gate-keeper during the complex assembly process of the oxygen-evolving centers in PSII.