Semin Boris K, Ghirardi Maria L, Seibert Michael
Basic Sciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, USA.
Biochemistry. 2002 May 7;41(18):5854-64. doi: 10.1021/bi0200054.
The donation of electrons by Mn(II) and Fe(II) to Y(Z*) through the high-affinity (HA(Z)) site in Mn-depleted photosystem II (PSII) membranes has been studied by flash-probe fluorescence yield measurements. Mn(II) and Fe(II) donate electrons to Y(Z*) with about the same efficiency, saturating this reaction at the same concentration (ca. 5 microM). However, following a short incubation of the membranes with 5 microM Fe(II), but not with Mn(II) in room light, added Mn(II) or Fe(II) can no longer be photooxidized by Y(Z)(). This blocking effect is caused by specifically bound, photooxidized Fe [> or =Fe(III)] and is accompanied by a delay in the fluorescence yield decay kinetics attributed to the slowing down of the charge recombination rate between Q(a-) and Y(Z). Exogenously added Fe(III), on the other hand, does not donate electrons to Y(Z*), does not block the donation of electrons by added Mn(II) and Fe(II), and does not change the kinetics of the decay of the fluorescence yield. These results demonstrate that the light-dependent oxidation of Fe(II) by Y(Z*) creates an Fe species that binds at the HA(Z) site and causes the blocking effect. The pH dependence of Mn(II) electron donation to Y(Z*) via the HA(Z) site and of the Fe-blocking effect is different. These results, together with sequence homologies between the C-terminal ends of the D1 and D2 polypeptides of the PSII reaction center and several diiron-oxo enzymes, suggest the involvement of two or perhaps more (to an upper limit of four to five) bound iron cations per reaction center of PSII in the blocking effect. Similarities in the interaction of Fe(II) and Mn(II) with the HA(Z) Mn site of PSII during the initial steps of the photoactivation process are discussed. The Fe-blocking effect was also used to investigate the relationship between the HA(Z) Mn site and the HA sites on PSII for diphenylcarbazide (DPC) and NH2OH oxidation. Blocking of the HA(Z) site with specifically bound Fe leads to the total inhibition of electron donation to Y(Z*) by DPC. Since DPC and Mn(II) donation to PSII is noncompetitive [Preston, C., and Seibert, M. (1991) Biochemistry 30, 9615-9624], the Fe bound to the HA(Z) site can also block the DPC donation site. On the other hand, electron donation by NH2OH to PSII still occurs in Fe-blocked membranes. Since hydroxylamine does not reduce the Fe [> or =Fe(III)] specifically bound to the HA(Z) site, NH2OH must donate to Y(Z*) through its own site or directly to P680+.
通过闪光探针荧光产率测量，研究了在缺锰的光系统II（PSII）膜中，Mn(II)和Fe(II)通过高亲和力（HA(Z)）位点向Y(Z*)供电子的情况。Mn(II)和Fe(II)以大致相同的效率向Y(Z*)供电子，在相同浓度（约5 microM）时使该反应达到饱和。然而，在室温光照下，将膜与5 microM Fe(II)短暂孵育后（但与Mn(II)孵育则不会），添加的Mn(II)或Fe(II)不再能被Y(Z)()光氧化。这种阻断效应是由特异性结合的光氧化Fe [≥Fe(III)]引起的，并且伴随着荧光产率衰减动力学的延迟，这归因于Q(a-)与Y(Z)之间电荷复合速率的减慢。另一方面，外源添加的Fe(III)不会向Y(Z*)供电子，不会阻断添加的Mn(II)和Fe(II)的电子供体作用，也不会改变荧光产率衰减的动力学。这些结果表明，Y(Z*)对Fe(II)的光依赖性氧化产生了一种在HA(Z)位点结合并导致阻断效应的Fe物种。Mn(II)通过HA(Z)位点向Y(Z*)供电子以及Fe阻断效应的pH依赖性是不同的。这些结果，连同PSII反应中心D1和D2多肽C末端之间的序列同源性以及几种双铁氧酶，表明PSII每个反应中心有两个或可能更多（上限为四到五个）结合的铁阳离子参与了阻断效应。讨论了在光激活过程的初始步骤中，Fe(II)和Mn(II)与PSII的HA(Z) Mn位点相互作用的相似性。Fe阻断效应还被用于研究HA(Z) Mn位点与PSII上用于二苯基卡巴腙（DPC）和NH2OH氧化的HA位点之间的关系。用特异性结合的Fe阻断HA(Z)位点会导致DPC向Y(Z*)供电子的完全抑制。由于DPC和Mn(II)向PSII的供电子是非竞争性的[普雷斯顿，C.，和塞伯特，M.（1991年）《生物化学》30，9615 - 9624]，结合在HA(Z)位点的Fe也可以阻断DPC的供电子位点。另一方面，在Fe阻断的膜中，NH2OH向PSII的供电子仍然发生。由于羟胺不会还原特异性结合在HA(Z)位点的Fe [≥Fe(III)]，NH2OH必定通过其自身的位点或直接向P680+供电子。
