Decay products of the S(3) state of the oxygen-evolving complex of photosystem II at cryogenic temperatures. Pathways to the formation of the S = 7/2 S(2) state configuration.

The water-oxidizing complex of photosystem II cycles through five oxidation states, denoted S(i)() (i = 0-4), during water oxidation to molecular oxygen, which appears at the (transient) S(4) state. The recent detection of bimodal EPR signals from the S(3) state [Matsukawa, T., Mino, H., Yoneda, D., Kawamori, A. (1999) Biochemistry 38, 4072-4077] has drawn significant attention to this critical state. An interesting property of the S(3) state is the sensitivity to near-IR (NIR) light excitation. Excitation of the S(3) state by near-IR light at cryogenic temperatures induces among other signals a derivative-shaped EPR signal at g= 5 [Ioannidis, N., and Petrouleas, V. (2000) Biochemistry 39, 5246-5254]. The signal bears unexpected similarities to a signal observed earlier in samples that had undergone multiple turnovers and subsequently had been stored at 77 K for a week or longer [Nugent, J. H. A., Turconi, S., and Evans, M. C. W. (1997) Biochemistry 36, 7086-7096]. Recently, both signals were assigned to an S = 7/2 configuration of the Mn cluster [Sanakis, Y., Ioannidis, N., Sioros, G., and Petrouleas, V. (2001) J. Am. Chem. Soc. 123, 10766-10767]. In the present study, we employ bimodal EPR spectroscopy to investigate the pathways of formation of this unusual state. The following observations are made: (i) The g = 5 signal evolves in apparent correlation with the diminution of the S(3) state signals during the slow (tens of hours to several days range) charge recombination of S(3) with Q(A)(-) at 77 K. The tyrosyl radical D* competes with S(3) for recombination with Q(A)(-), the functional redox couple at cryogenic temperatures inferred to be D*/D(-). Transfer to -50 degrees C and above results in the relaxation of the g = 5 to the multiline and g = 4.1 signals of the normal S(2) state. (ii) The transition of S(3) to the state responsible for the g = 5 signal can be reversed by visible light illumination directly at -30 degrees C or by illumination at 4.2 K followed by brief (2 min) transfer to -50 degrees C in the dark. The latter step is required in order to overcome an apparent thermal activation barrier (charge recombination appears to be faster than forward electron transfer at 4.2 K). (iii) The "g = 5" state can be reached in a few tens of minutes at 4.2 K by near-IR light excitation of the S(3) state. This effect is attributed to the transfer of the positive hole from the Mn cluster to a radical (probably tyr Z), which recombines much faster than the Mn cluster with Q(A)(-). (iv) The above properties strongly support the assignment of the configuration responsible for the g = 5 signal to a modified S(2) state, denoted S(2)'. Evidence supporting the assignment of the S(2)' to a proton-deficient S(2) configuration is provided by the observation that the spectrum of S(2) at pH 8.1 (obtained by illumination of the S(1) state at -30 degrees C) contains a g = 5 contribution.