Fromwald S, Zoder R, Wastyn M, Lübben M, Peschek G A
Molecular Bioenergetics Group, Institute of Physical Chemistry, University of Vienna, Wien, A-1090, Austria.
Arch Biochem Biophys. 1999 Jul 1;367(1):122-8. doi: 10.1006/abbi.1999.1236.
The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 microliters packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase high-performance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a3 and o3, both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa3-type cytochrome c oxidase. The heme D, on the other hand, seems to form a spectrally distinguished, yet kinetically ill-defined hemoprotein complex which does not qualify as a fully functional d-type terminal oxidase on our (wild-type) cyanobacteria even after growth under semianaerobic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under dinitrogen-fixing conditions did not change this situation. Thus, we are left with (wild-type) cyanobacteria forming an unbranched respiratory chain with only a single type of terminal oxidase protein, viz. the known aa3-type cytochrome c oxidase. This oxidase, however, may incorporate different prosthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox environment. In particular, however, conditions for expression of the two quinol oxidases potentially and additionally coded for by the genome of, e. g., Synechocystis sp. PCC6803 (see http://www.kazusa.or.jp/cyano), have not yet been found.
将蓝细菌集胞藻（聚球藻属PCC6301）、聚球藻属PCC6803、鱼腥藻属PCC 7120和念珠藻属PCC8009在低氧张力下进行光自养培养，培养基中的硫酸盐被硫代硫酸盐取代，硝酸盐被铵取代，分别作为硫源和氮源。此外，鱼腥藻和念珠藻在无化合态氮的培养基中于固氮条件下培养。从对数生长后期的细胞（培养密度相当于每毫升约3微升压实细胞）中分离出膜；根据既定程序分离并纯化细胞质膜和类囊体膜。从膜中提取酸不稳定血红素，并进行反相高效液相色谱分析。对分离出的血红素进行光谱分析，并与标准品比较进行鉴定。除了血红素B、A和O（后者仅在半厌氧条件下诱导产生）外，用硫代硫酸盐和铵取代含氧阴离子硫酸盐和硝酸盐后，在膜及其提取物中出现了光谱上可区分的血红素D。然而，对膜结合血红素D的光谱和动力学研究有力地反驳了其与氧气或一氧化碳发生任何反应的观点。对膜结合呼吸氧化酶进行的动力学测量表明，只有两种具有动力学活性的末端氧化酶，即a3和o3，显然都与单一类型的脱辅基蛋白相关，即已知蓝细菌aa3型细胞色素c氧化酶的亚基I。另一方面，血红素D似乎形成了一种光谱上有区别但动力学上不明确的血红素蛋白复合物，即使在半厌氧假还原条件下生长后，在我们的（野生型）蓝细菌中也不符合作为完全功能性d型末端氧化酶的条件。在固氮条件下（鱼腥藻和念珠藻的）生长也没有改变这种情况。因此，我们得到的（野生型）蓝细菌形成了一条无分支的呼吸链，只有单一类型的末端氧化酶蛋白，即已知的aa3型细胞色素c氧化酶。然而，这种氧化酶在“血红素混杂”的意义上可能包含不同的辅基（血红素）基团。不同血红素基团的生物合成似乎对周围的氧化还原环境有反应。特别是，例如聚球藻属PCC6803基因组可能额外编码的两种喹啉氧化酶的表达条件（见http://www.kazusa.or.jp/cyano）尚未找到。
