RESUMEN
At the request of the organizer of this special edition, we have attempted to do several things in this manuscript: (1) we present a mini-review of recent, selected, works on the light-induced inorganic biogenesis (photoactivation), composition and structure of the inorganic core responsible for photosynthetic water oxidation; (2) we summarize a new proposal for the evolutionary origin of the water oxidation catalyst which postulates a key role for bicarbonate in formation of the inorganic core; (3) we summarize published studies and present new results on what has been learned from studies of 'inorganic mutants' in which the endogenous cofactors (Mn(n+), Ca2+, Cl-) are substituted; (4) the first DeltapH changes measured during the photoactivation process are reported and used to develop a model for the stepwise photo-assembly process; (5) a comparative analysis is given of data in the literature on the kinetics of substrate water exchange and peroxide binding/dismutation which support a mechanistic model for water oxidation in general; (6) we discuss alternative interpretations of data in the literature with a view to forecast new avenues where progress is needed.
Asunto(s)
Oxígeno/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Agua/química , Sitios de Unión , Calcio/química , Catalasa/química , Cloruros/química , Evolución Molecular , Manganeso/química , Espectrometría de Masas , Modelos Químicos , Oxidación-Reducción , Fotosíntesis , Factores de TiempoRESUMEN
The process of photoactivation, the assembly of a functional water-oxidizing complex (WOC) from the apoproteins of photosystem II of higher plants and inorganic cofactors (Mn2+, Ca2+, and Cl-), was known from earlier works to be a two-step kinetic process, requiring two light-induced processes separated by a slower dark period. However, these steps had not been directly resolved in any kinetic experiment, until development of an ultrasensitive polarographic O2 electrode and synthesis of an improved chelator for cofactor removal allowed direct kinetic resolution of the first pre-steady state intermediate [Ananyev, G. M. & Dismukes, G. C. (1996a) Biochemistry 35, 4102-4109]. Herein, the dependence of the rates of each of the first two light steps and the dark step of photoactivation was directly determined in spinach PSII membranes over a range of calcium and manganese concentrations at least 10-fold lower than those possible using commercial O2 electrodes. The following results were obtained. (1) One Mn2+ ion binds and is photooxidized to Mn3+ at a high-affinity site, forming the first light-induced intermediate, IM1. Formation of IM1 is coupled to the dissociation of a bound Ca2+ ion either located in the Mn site or coupled to it. (2) The inhibition constant for Ca2+ dissociation from this site is equal to 1.5 mM. (3) The dissociation constant of Mn2+ at this high-affinity site is equal to 8 microM at the optimum calcium concentration for O2-evolving activity of 8 mM, in agreement with the high-affinity site for electron donation to PSII. (4) Prior to the next photolytic step, one Ca2+ ion must bind at its effector site so that stable photooxidation of a second Mn2+ ion can occur, forming the second light-induced intermediate, IM2. This dark process is the rate-determining step. (5) The Michaelis constant for recovery of O2 evolution by Ca2+ binding at this effector site (Km) is equal to 1.4 mM, a value that is the same as that measured for the calcium requirement for O2 evolution in intact PSII. (6) The low quantum yield for the formation of IM2 from IM1 increases linearly with the duration of the dark period up to the longest period we could examine (10 s). Accordingly, the rate limitation in the second photolytic step originates from a slow calcium-induced dark rearrangement of the first intermediate, IM1, which we propose to be a protein conformational change that allows stable binding of the next Mn2+ ion. We further propose that the single Ca2+ ion which is required for assembly of the Mn4 cluster is equivalent to the Ca2+ ion which functions at the "gatekeeper" site in intact O2-evolving centers, where it plays a role in limiting substrate access to the Mn4 cluster [Sivaraja, M., et al. (1989) Biochemistry 28, 9459-9464; Tso, J., et al., (1991) Biochemistry 30, 4734-4739]. A molecular model for photoactivation is proposed and discussed.