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 language | English |
|---|---|
| Pages (from-to) | 14608-14617 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 35 |
| Issue number | 46 |
| DOIs | |
| Publication status | Published - Nov 19 1996 |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
Cite this
High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation : Proton equilibrium, cations, and electrostatics. / Ananyev, Gennady M.; Dismukes, G Charles.
In: Biochemistry, Vol. 35, No. 46, 19.11.1996, p. 14608-14617.Research output: Contribution to journal › Article
}
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.
UR - http://www.scopus.com/inward/record.url?scp=0030465649&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030465649&partnerID=8YFLogxK
U2 - 10.1021/bi960894t
DO - 10.1021/bi960894t
M3 - Article
C2 - 8931559
AN - SCOPUS:0030465649
VL - 35
SP - 14608
EP - 14617
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 46
ER -