Analysis of the oxidation-reduction potentials of recombinant ferredoxin-NADP(+) reductase from spinach chloroplasts

被引:35
作者
Corrado, ME
Aliverti, A
Zanetti, G
Mayhew, SG
机构
[1] NATL UNIV IRELAND UNIV COLL DUBLIN, DEPT BIOCHEM, BELFIELD 4, DUBLIN, IRELAND
[2] UNIV MILAN, DIPARTIMENTO FISIOL & BIOCHIM GEN, MILAN, ITALY
[3] UNIV MILAN, CTR INTERUNIV STUDIO MACROMOL INFORMAZ, MILAN, ITALY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1996年 / 239卷 / 03期
关键词
flavoprotein; FAD; redox potential; spinach ferredoxin-NADP(+) reductase; flavin hydroquinone anion;
D O I
10.1111/j.1432-1033.1996.0662u.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Midpoint oxidation-reduction potentials for the two-electron reduction of the bound FAD in spinach ferredoxin-NADP(+) reductase were measured by potentiometry (E(m) = -342 +/- 1 mV at pH 7 and 10 degrees C). They were used with the semiquinone formation constant, obtained by spectroscopic measurement of the semiquinone concentration. to calculate values for the redox potentials of the two one-electron steps in the reduction. The redox potential for the oxidized enzyme/enzyme semiquinone couple (E(OX/SQ)) at pH 7 is -350 +/- 2 mV (10 degrees) while the value for the enzyme semiquinone/enzyme hydroquinone couple (E(SQ/HQ)) under the same conditions is -335 +/- 1 mV. These values correspond to a semiquinone formation constant of 0.55. Measurement of the effects of pH on the potentials showed that E(OX/SQ) varies linearly with pH (slope -46 +/- 4 mV), while E(SQ/HQ) is independent of pH at high pH values, but below about pH 7.5 the potential becomes less negative with decreasing pH, indicating that there isa redox-linked protonation of the fully reduced enzyme (pK(a) = 7.2, 10 degrees C). The absorption spectrum of the fully reduced enzyme was found to depend on pH with the changes giving a calculated pK(a) of 7.5 (at 15 degrees C). The spectrum at high pH is similar to that of the anionic form of free flavin hydroquinone. The observations suggest that at physiological pH, the enzyme FAD cycles between the three redox states: oxidized, neutral semiquinone and hydroquinone anion.
引用
收藏
页码:662 / 667
页数:6
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