Porra R J, Schäfer W, Cmiel E, Katheder I, Scheer H
Botanisches Institut, Universität, München, Germany.
Eur J Biochem. 1994 Jan 15;219(1-2):671-9. doi: 10.1111/j.1432-1033.1994.tb19983.x.
The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H2(18)O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlorophyll b-7(1)-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either H2(18)O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 13(1)-oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599-602]. The incorporation of isotope led to as much as 77% enrichment of the 13(1)-oxo group of chlorophyll a: assuming identical incorporation into the 13(1) oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The high enrichment using 18O2, and the absence of labelling by H2(18)O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme.
叶绿素b甲酰基的形成机制长期以来一直不清楚，但在本文中，通过对从黑暗生长的植物上切下的黄化玉米叶片进行照光绿化，在H₂¹⁸O或¹⁸O₂存在的白光下进行处理，并通过质谱分析新合成的叶绿素，确定了高等植物中叶绿素b的7-甲酰基氧的来源。为了尽量减少细胞中未标记的水与叶绿素b-7(1)-偕二醇（水合物）可逆形成导致7-甲酰基取代基上¹⁸O标记可能的损失，在用含NaBH₄的甲醇萃取过程中，甲酰基被还原为羟甲基：在此快速还原萃取过程中叶绿素a保持不变。从在H₂¹⁸O或¹⁸O₂存在下绿化的叶片中提取的叶绿素a和[7-羟甲基]-叶绿素b的质谱显示，¹⁸O仅从分子氧中掺入，但两种叶绿素中都有掺入：质谱与分子氧不仅为掺入叶绿素b的7-甲酰基提供一个氧原子，也为已充分证明的掺入叶绿素a和b的13(1)-氧代基团的情况相符[见Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599 - 602]。同位素的掺入导致叶绿素a的13(1)-氧代基团富集高达77%：假设叶绿素b的13(1)氧中掺入情况相同，那么7-甲酰基氧的富集高达93%。如在3-(3,4-二氯苯基)-1,1-二甲基脲存在下的绿化实验所示，光合作用产生的来自未标记水的氧重新掺入导致的同位素稀释可忽略不计。使用¹⁸O₂时的高富集以及H₂¹⁸O未标记，明确表明分子氧是高等植物中叶绿素b的7-甲酰基氧的唯一前体，并强烈暗示存在一条涉及加氧酶型酶参与的叶绿素b甲酰基形成的单一途径。
