Ponamarev M V, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
Biochemistry. 1998 Dec 8;37(49):17199-208. doi: 10.1021/bi981814j.
The 1.96 A structure of turnip cytochrome f revealed a linear internal chain of H2O molecules with the oxygen atoms of the chain having occupancies and "B" factors comparable to those of neighboring atoms [Martinez et al. (1996) Protein Sci. 5, 1081-1092. ]. Four waters extend 11 A from the heme toward Lys66 on the cytochrome surface. All residues that contribute an atom to the 15 H-bonds of five internal H2O molecules are essentially conserved in 23 cytochrome sequences. With only Gln and Asn side chains involved in H-bonding, the water chain resembles a "proton wire". The function of the conserved H2O chain was tested through site-directed mutagenesis of these Asn and Gln residues. Four of the five conserved Asn/Gln residues were changed in six mutants generated in the green alga, Chlamydomonas reinhardtii. Except for the N168F mutant, all grew photosynthetically. Although the rates of oxidation of cyt f oxidation and of reduction of cyt b6 (5-6 ms in the wild type) were not significantly affected, the rates of cyt f reduction and generation of the slow electrochromic band shift (Deltapsis) were markedly decreased, the half-times increasing to as much as 38 and 18 ms, respectively. Thus, in these mutants, reduction of cyt b6 reduction clearly precedes that of cyt f. Retardation of Deltapsis in the absence of an observable change in the rate of cyt b6 reduction implied that the rate of H+ translocation decreased in the mutants, and electron transfer was concomitantly retarded, most likely between the ISP and cyt f. The following was concluded: (i) proton and electron transfer are coupled in reduction of cyt f, and the cyt f water chain functions in H+ transfer; (ii) reduction of the high- and low-potential chains in the b6f complex is not concerted in the water chain mutants; and (iii) quinol deprotonation and electron transfer from reduced quinone are initiated by an early event, probably the movement of the ISP triggered by oxidation of cyt f.
芜菁细胞色素f的1.96 Å结构揭示了一条由水分子组成的线性内部链，链中的氧原子占有率和“B”因子与相邻原子相当[Martinez等人（1996年），《蛋白质科学》5，1081 - 1092页]。四个水分子从血红素向细胞色素表面的赖氨酸66延伸11 Å。对五个内部水分子的15个氢键有贡献的所有残基在23个细胞色素序列中基本保守。由于只有谷氨酰胺和天冬酰胺侧链参与氢键形成，水链类似于一条“质子线”。通过对这些天冬酰胺和谷氨酰胺残基进行定点诱变来测试保守水链的功能。在莱茵衣藻中产生的六个突变体中，五个保守的天冬酰胺/谷氨酰胺残基中的四个发生了变化。除了N168F突变体，其他所有突变体都能进行光合作用。虽然细胞色素f氧化的速率和细胞色素b6还原的速率（野生型中为5 - 6毫秒）没有受到显著影响，但细胞色素f还原的速率和慢电致变色带移（Δψs）的产生速率明显降低，半衰期分别增加到多达38毫秒和18毫秒。因此，在这些突变体中，细胞色素b6的还原明显先于细胞色素f的还原。在细胞色素b6还原速率没有明显变化的情况下，Δψs的延迟表明突变体中H⁺转运速率降低，电子传递也随之延迟，最有可能发生在铁硫蛋白和细胞色素f之间。得出以下结论：（i）在细胞色素f的还原过程中质子和电子传递是偶联的，细胞色素f水链在H⁺传递中起作用；（ii）在水链突变体中，b6f复合物中高电位和低电位链的还原不是协同进行的；（iii）醌去质子化和还原醌的电子传递由一个早期事件引发，可能是细胞色素f氧化触发的铁硫蛋白的移动。
