High-Resolution Kinetic Studies of the Reassembly of the Tetra-Manganese Cluster of Photosynthetic Water Oxidation: Proton Equilibrium, Cations, and Electrostatics ² Gennady M. Ananyev and G. Charles Dismukes* Hoyt Laboratory, Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544 ReceiVed April 15, 1996; ReVised Manuscript ReceiVed September 9, 1996 X ABSTRACT: The kinetics of pulsed-light photoactivation, the light-induced reassembly of the water-oxidizing complex (WOC) of PSII in the presence of essential inorganic cofactors, has been studied using two improvements: a new efficient chelator, N,N,N′,N′-tetrapropionato-1,3-bis(aminomethyl)benzene (TPDBA), for complete extraction of {Mn 4 } and Ca 2+ and an ultrasensitive polarographic cell for O 2 detection [Ananyev, G. M., & Dismukes, G. C. (1996) Biochemistry 35, 4102-4109]. Measurements have been made of the initial half-time, t 1/2 (sum of the lag time for formation of the first intermediate, IM 1 , plus the half-time for formation of the second intermediate, IM 2 ), and the steady-state yield, Y ss , for recovery of O 2 evolution (proportional to the number of active centers). The following conclusions have been reached: (1) cations (Ca 2+ , Mg 2+ , and Na + ) slow the rate of photoactivation, even though Ca 2+ is essential for activity. Two distinct mechanisms appear to be involved: binding to one or both of the first two Mn 2+ -specific sites and screening of negative charges on apo-WOC that are responsible for concentrating Mn 2+ ions by electrostatic steering; (2) the Michaelis constant for the calcium requirement for Y ss at sufficiently low Mn 2+ concentrations (8 µM) that competition at the calcium site does not occur is K m ) 1.4 mM. Numerically, K m is the same for reactivation of O 2 evolution in Ca-depleted PSII membranes which retain four Mn ions; (3) in the absence of Ca 2+ but in the presence of saturating amounts of Mn 2+ (8 Mn/apo-WOC) and Cl - (35 mM) assembly of a stable tetra-Mn cluster occurs neither under illumination nor in the dark after subsequent addition of CaCl 2 . However, in the presence of suboptimal concentrations of calcium required for maximum Y ss , calcium-dependent assembly of stable yet inactive clusters occurs in the light; (4) protons in equilibrium with the buffer greatly increase the half-time 3-fold between pH 6.75 and 5.4, indicating ionization of one or more protons from the first photo-oxidized intermediate formed prior to the rate-limiting step (photo-oxidation of the second Mn 2+ ); (5) the lipophilic membrane soluble anion tetraphenylboron (TPB - ), a known reductant of intact WOC, increases the half-time 2.5- fold (e40 µM) and paradoxically stimulates Y ss by 50% at 20 µM concentration. These results suggest that TPB - increases the local concentration of Mn 2+ adjacent to apo-WOC (Y ss increase), while also reducing the S 2 and S 3 states of the intact WOC at higher concentrations (t 1/2 increase). The effects of anions and cations indicates that overcoming the surface potential of the membrane/protein PSII complex may play an important role in the kinetics of reassembly of the {Mn 4 } cluster; (6) the ratio Y 4 /Y 3 in the kinetics of O 2 evolution from a series of single-turnover flashes, a ratio that typically reflects the probability of misses (R), grows noticeably larger with increasing extent of recovery of O 2 evolving activity and also with increase in the amount of Mn 2+ , indicating competition between substrate water and excess Mn 2+ for reduction of the functional {Mn 4 } cluster. On the basis of these results, we extend the model for photoactivation to include the antagonistic effects of H + and Ca 2+ in the formation of the first two intermediates. Unlike several stable inorganic clusters observed in biology which undergo spontaneous self-assembly, the WOC 1 pos- sesses an intrinsically unstable core. The tetra-Mn-Ca active center of the WOC of PSII can be reversibly reassembled only in the presence of the apo-PSII membrane/protein complex by a light-driven process called photoactivation (Radmer & Cheniae, 1977; Cheniae, 1980). A few kinetic models for the photoactivation process have been proposed, all in agreement on the formation of a metastable intermedi- ate in the rate-limiting step which requires Mn 2+ and both light and dark steps to reach (Radmer & Cheniae, 1971; Tamura & Cheniae, 1987; Tamura et al., 1989; Miller & Brudvig, 1989; Blubaugh & Cheniae, 1992). However, supporting evidence that would identify the molecular structures, oxidation states, and chemical reactivities of the proposed intermediates is very limited. On the basis of steady-state kinetic and biochemical studies Cheniae’s group (Tamura & Cheniae, 1987; Blubaugh & ² This work was supported by the National Institutes of Health (Grant GM39932). * To whom correspondence should be addressed. FAX: (609) 258- 1980. E-mail: dismukes@chemvax.princeton.edu. X Abstract published in AdVance ACS Abstracts, October 15, 1996. 1 Abbreviations: Chl, chlorophyll; Bis-Tris, bis(2-hydroxyethyl)- iminotris(hydroxymethyl)methane; LED, light-emitting diode; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethane- sulfonic acid; P680, primary electron donor; PSII, photosystem II; Pheo, primary pheophytine electron acceptor of PSII; QA and QB, primary and secondary plastoquinone electron acceptors; RC, reaction center; t1/2, half-time kinetics of pulsed-light photoactivation of O2 evolution; TPB, tetraphenylboron; TPDBA, N,N,N′,N′-tetrapropionato-1,3-bis- (aminomethyl)benzene; VO2 , maximal rate of O2 evolution at saturated continuous light after pulsed-light activation; Yss, steady-state level kinetics of pulsed-light photoactivation of O2 evolution; Yz, redox-active tyrosine-161 of the D1 polypeptide; WOC, water-oxidizing complex. 14608 Biochemistry 1996, 35, 14608-14617 S0006-2960(96)00894-X CCC: $12.00 © 1996 American Chemical Society