EPR Study of the Oxygen Evolving Complex in His-Tagged Photosystem II from the Cyanobacterium Synechococcus elongatus

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Abstract
The Mn4-cluster and the cytochrome c550 in histidine-tagged photosystem II (PSII) from Synechococcus elongatus were studied using electron paramagnetic resonance (EPR) spectroscopy. The EPR signals associated with the S0-state (spin = 1/2) and the S2-state (spin = 1/2 and IR-induced spin = 5/2 state) were essentially identical to those detected in the non-His-tagged strain. The EPR signals from the S3-state, not previously reported in cyanobacteria, were detectable both using perpendicular (at g = 10) and parallel (at g = 14) polarization EPR, and these signals are similar to those found in plant PSII. In the S3-state, near-infrared illumination at 50 K induced a 176-G-wide split signal at g = 2 and signals at g = 5.20 and g = 1.51. These signals differ slightly from those reported in plant PSII [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246−5254]. In accordance with the cited work, the split signal presumably reflects a radical interacting with the Mn4-cluster in a fraction of centers, while the g = 5.20 and g = 1.51 signals are tentatively attributed to a high-spin state of the Mn4-cluster with zero field splitting parameters different from those in plant PSII, reflecting minor changes in the environment of the Mn4-cluster. Biochemical modifications (Sr2+/Ca2+ substitution, acetate and NH3 treatments) were also investigated. In Sr2+-reconstituted PSII, in addition to the expected modified S2 multiline signal, a signal at g = 5.2 was present instead of the g ≈ 4 signal seen in plant PSII. In NH3-treated samples, in addition to the expected modified S2-multiline signal, a g ≈ 4 signal was detected in a small proportion of the reaction centers. This is of note since g ≈ 4 spectra arising from the Mn4-cluster in the S2 state have not yet been published in cyanobacterial PSII. The detection of modified S3-signals in both perpendicular (at g = 7.5) and parallel (at g = 12) polarization EPR from NH3-treated PSII indicate that NH3 is still bound in the S3-state. The acetate-treated PSII behaves essentially as in plant PSII. A study using oriented samples indicated that the heme plane of the oxidized low spin Cytc550 was perpendicular to the plane of the membrane.