High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation

Proton equilibrium, cations, and electrostatics

Gennady M. Ananyev, G Charles Dismukes

Research output: Contribution to journalArticle

48 Citations (Scopus)

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 {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.

Original languageEnglish
Pages (from-to)14608-14617
Number of pages10
JournalBiochemistry
Volume35
Issue number46
DOIs
Publication statusPublished - Nov 19 1996

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Manganese
Static Electricity
Cations
Protons
Electrostatics
Oxidation
Kinetics
Water
Tetraphenylborate
Calcium
Light
Anions
Ions
Membranes
Recovery
Biochemistry
Photooxidation
Reducing Agents
Chelating Agents
Lighting

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{d53f3ab5c2e24f31b3c1d2ffca2783d1,
title = "High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: Proton equilibrium, cations, and electrostatics",
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 {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.",
author = "Ananyev, {Gennady M.} and Dismukes, {G Charles}",
year = "1996",
month = "11",
day = "19",
doi = "10.1021/bi960894t",
language = "English",
volume = "35",
pages = "14608--14617",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "46",

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TY - JOUR

T1 - High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation

T2 - Proton equilibrium, cations, and electrostatics

AU - Ananyev, Gennady M.

AU - Dismukes, G Charles

PY - 1996/11/19

Y1 - 1996/11/19

N2 - 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.

AB - 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.

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