High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: Proton equilibrium, cations, and electrostatics

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 {Mn₄} and Ca²⁺ and an ultrasensitive polarographic cell for O₂ detection [Ananyev, G. M., and 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, IM₁, plus the half-time for formation of the second intermediate. IM₂), and the steady-state yield, Y(ss), recovery of O₂ evolution (proportional to the number of active centers). The following conclusions have been reached: (1) cations (Ca²⁺, Mg²⁺, and Na⁺) slow the rate of photoactivation, even though Ca²⁺ is essential for activity. Two distinct mechanisms appear to be involved: binding to one or both of the first two Mn²⁺-specific sites and screening of negative charges on apo-WOC that are responsible for concentrating Mn²⁺ ions by electrostatic steering: (2) the Michaelis constant for the calcium requirement for Y(ss) at sufficiently low Mn²⁺ 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 O₂ evolution in Ca-depleted PSII membranes which retain four Mn ions: (3) in the absence of Ca²⁺ but in the presence of saturating amounts of Mn²⁺ (8 Mn/apo-WOC) and Cl^- (35 mM) assembly of a stable tetra- Mn cluster occurs neither trader illumination nor in the dark after subsequent addition of CaCl₂. 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 Mn²⁺); (5) the lipophilic membrane soluble anion tetraphenylboron (TPB^-), a known reductant of intact WOC, increases the half-time 2.5-fold (≤40 μM) and paradoxically stimulates Y(ss) by 50% at 20 μM concentration. These results suggest that TPB^- increases the local concentration of Mn²⁺ adjacent to apo-WOC (Y(ss) increase), while also reducing the S₂ and S₃ 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 {Mn₄} cluster; (6) the ratio Y₄/Y₃ in the kinetics of O₂ evolution from u series of single-turnover flashes, a ratio that typically reflects the probability of misses (α), grows noticeably larger with increasing extent of recovery of O₂ evolving activity and also with increase in the amount of Mn²⁺, indicating competition between substrate water and excess Mn²⁺ for reduction of the functional {Mn₄} cluster. On the basis of these results, we extend the model for photoactivation to include the antagonistic effects of H⁺ and Ca²⁺ in the formation of the first two intermediates.