二氧化碳和臭氧浓度升高对四季竹矿质养分含量和运输的影响

为阐明CO_2和O_3浓度升高对竹子矿质养分含量和运输的影响,以2年生四季竹(Oligostachyum lubricum)为试材,采用开顶式气室(OTCs)设置了环境背景大气[CK,(40±5)nmol·mol~(-1) O_3,(360±20)μmol·mol~(-1) CO_2]、O_3浓度升高[EO,(100±10)nmol·mol~(-1) O_3,(360±20)μmol·mol~(-1) CO_2]、CO_2浓度升高[EC,(40±5)nmol·mol~(-1) O_3,(700±35)μmol·mol~(-1) CO_2]、CO_2和O_3浓度复合升高[EOEC,(100±10)nmol·mol~(-1) O_3,(700±35)μmol·mol~(-1) CO_2]4个处理,测定了竹叶、竹枝、竹秆和竹根的Na+、Fe(~(2+,3+)、Ca~(2+)和Mg~(2+)含量,并分析了矿质营养在器官间的转运情况。结果显示:与CK比较,EO处理显著降低了四季竹植株体内Na+和Fe(~(2+,3+)含量,特别是竹根和竹叶,同时也明显降低Na+和Fe(~(2+,3+)器官间的运输能力。EC处理显著降低了四季竹植株体内Na+含量,而未改变其他矿质元素含量,但其器官中的分配格局和运输能力发生变化,尤其是竹枝向竹叶运输Ca~(2+)和Mg~(2+)的能力增强。EOEC处理显著降低了四季竹植株体内Na+含量,但显著提高了Fe(~(2+,3+)、Ca~(2+)和Mg~(2+)含量及其向上运输能力。研究表明,O_3浓度升高降低了四季竹植株体内矿质养分含量和器官间养分运输能力,在一定程度上影响四季竹的正常生长;CO_2浓度升高通过提高Ca~(2+)和Mg~(2+)向光合器官叶片的运输能力,促进四季竹生长;CO_2和O_3浓度升高复合作用能够通过提高四季竹光合器官竹叶中Fe(~(2+,3+)、Ca~(2+)和Mg~(2+)含量及其向光合器官叶片的运输能力,以维持体内矿质养分元素的平衡,提高四季竹对高浓度CO_2和O_3浓度复合环境下的适应能力。     

盐胁迫对四季竹细胞膜透性和矿质离子吸收、运输和分配的影响

采用盆栽控制试验,研究了土壤不同NaCl浓度(0(CK)、1‰、2‰、3‰、4‰、5‰和6‰)处理45 d对四季竹叶片脱落率和细胞膜透性以及立竹器官K+、Na+、Ca2+和Cl-等矿质离子的吸收、运输和分配的影响.结果表明,1‰～2‰NaCl处理对四季竹叶片脱落率和离子渗漏率无显著影响,3‰～6‰ NaCl处理显著提高了叶片脱落率和离子渗漏率,四季竹的盐胁迫伤害随土壤盐浓度的增大而加剧.随着Na+、Cl-在四季竹立竹各器官中的显著增加,竹根、竹秆、竹枝K+含量逐渐下降,Ca2+含量变化较小,并且K+、Ca2+在竹根、竹秆中的向上选择性运输能力逐渐减弱.由于竹叶在低浓度(1‰～2‰)和高浓度(3‰～6‰)盐胁迫下分别对Ca2+和K+具有较高的选择性吸收能力,随盐浓度的增大,竹叶K+含量迅速升高,Ca2+含量先升高后下降,这对维持竹叶的营养平衡和正常生长具有重要意义.3‰～6‰NaCl处理时,Na+、Cl-在竹叶中的浓度显著高于立竹其他器官,不仅降低了竹叶的渗透势,有利于水分的向上运输,而且四季竹还可以通过叶片脱落的方式降低体内的盐分含量,减轻盐离子毒害.     

外源EBR对NaCl胁迫下燕麦幼苗无机离子吸收、运输和分配的影响

为了探讨外源2,4-表油菜素内酯(2,4-epibrassinolide,EBR)诱导燕麦(Avena sativa L.)幼苗抗盐性的效果及其生理调节机制,以"青引2号"和"加燕2号"燕麦为材料,研究NaCl胁迫下施用外源EBR对燕麦幼苗无机离子吸收、运输和分配的影响。结果表明:100mmol·L-1NaCl胁迫下,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+、Cl-含量均显著升高,对阳离子的吸收产生了拮抗作用,导致燕麦幼苗叶片和根系中的K+、Ca2+、Mg2+、Mn2+、Fe2+、Zn2+、Cu2+含量显著降低,离子稳态平衡被打破;100 mmol·L-1NaCl胁迫下,施用0.01μmol·L-1外源EBR后,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+和Cl-含量显著降低,促进了燕麦幼苗根系对K+、Ca2+、Mg2+、Fe2+、Mn2+、Cu2+和Zn2+的吸收,叶片和根系中K+/Na+、Cl-/Na+、Ca2+/Na+、Mg2+/Na+、Fe2+/Na+、Mn2+/Na+、Cu2+/Na+和Zn2+/Na+显著升高,并且有效调控燕麦幼苗体内无机离子的运输比和阳离子的运输选择性比率,离子稳态重新达到平衡状态;说明外源EBR能够缓解NaCl胁迫下Na+和Cl-对燕麦幼苗所造成的离子毒害作用,有效调控燕麦幼苗对无机离子的选择性吸收、运输和分配,对维持燕麦幼苗体内的离子稳态平衡具有正向调控作用。     

外源EBR对NaCl胁迫下燕麦幼苗无机离子吸收、运输和分配的影响

为了探讨外源2,4-表油菜素内酯(2,4-epibrassinolide,EBR)诱导燕麦(Avena sativa L.)幼苗抗盐性的效果及其生理调节机制,以"青引2号"和"加燕2号"燕麦为材料,研究NaCl胁迫下施用外源EBR对燕麦幼苗无机离子吸收、运输和分配的影响。结果表明:100mmol·L-1NaCl胁迫下,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+、Cl-含量均显著升高,对阳离子的吸收产生了拮抗作用,导致燕麦幼苗叶片和根系中的K+、Ca2+、Mg2+、Mn2+、Fe2+、Zn2+、Cu2+含量显著降低,离子稳态平衡被打破; 100 mmol·L-1NaCl胁迫下,施用0.01μmol·L-1外源EBR后,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+和Cl-含量显著降低,促进了燕麦幼苗根系对K+、Ca2+、Mg2+、Fe2+、Mn2+、Cu2+和Zn2+的吸收,叶片和根系中K+/Na+、Cl-/Na+、Ca2+/Na+、Mg2+/Na+、Fe2+/Na+、Mn2+/Na+、Cu2+/Na+和Zn2+/Na+显著升高,并且有效调控燕麦幼苗体内无机离子的运输比和阳离子的运输选择性比率,离子稳态重新达到平衡状态;说明外源EBR能够缓解NaCl胁迫下Na+和Cl-对燕麦幼苗所造成的离子毒害作用,有效调控燕麦幼苗对无机离子的选择性吸收、运输和分配,对维持燕麦幼苗体内的离子稳态平衡具有正向调控作用。     

连续两个生长季大气CO2浓度升高对银杏希尔反应活力和叶绿体ATP酶活性的影响

近年来,随着温室气体浓度不断上升,有关CO2浓度升高对植物影响的研究已取得一定进展,但CO2浓度升高对植物光合作用的影响需要从生理生化水平上进一步深入的研究.以沈阳城市森林树种银杏(Ginkgo biloba L.)为研究对象,利用开顶式气室研究连续两个生长季大气CO2浓度升高对银杏光合特性的影响.结果表明,在大气CO2浓度为700μmol·mol-1条件下,与对照相比,第1个生长季CO2处理的银杏叶片净光合速率极显著增加(P<0.01),希尔反应活力极显著增大(P<0.01)、Ca2+/Mg2+-ATP酶活性显著(P<0.05)或极显著增强(P<0.01)、光合产物淀粉的含量极显著增多(P<0.01);第2生长季CO2处理的银杏叶片净光合速率显著增加(P<0.05),希尔反应活力在通气60d时极显著(P<0.01)增大,Ca2+/Mg2+-ATP酶活性在处理30d时显著降低(P<0.05),淀粉含量增多.与第1个生长季相比,第2个生长季CO2处理的银杏叶片净光合速率降低,希尔反应活力减小,Ca2+/Mg2+-ATP酶活性减弱,叶绿素含量增多,淀粉含量减少.试验中出现了光合适应现象.     

不同竹龄雷竹中硅及其他营养元素吸收和积累特征

在浙江省临安市的雷竹主产区,分别采集不同竹龄(1 ～4 a)和不同器官(叶、枝、秆)的雷竹样品,分析了Si和其他营养元素含量、吸收和积累特征,以及Si和其他营养元素之间的相互关系.结果表明:雷竹各器官中C含量的大小顺序为竹秆＞竹枝＞竹叶,Si、N、P、K、Ca、Mg、AI、Fe和Mn含量的大小顺序为竹叶＞竹枝＞竹秆.除Mn主要积累在竹叶中外,其他9种营养元素主要积累在1年生雷竹的秆中.3～4年生雷竹竹叶的Si平均含量为13.66g·kg-1.雷竹属于Si积累植物.随竹龄的增加,雷竹叶中的N、P、K和Mg含量减少,C、Al和Mn含量增加.雷竹对Si的吸收主要集中在第2年(57.1％),对N和K的吸收主要集中在前两年(67.7％～93.7％),此后N和K从植株体内流出,其流失量分别占总积累量的19.1％～39.1％.雷竹中Si与Ca、Al、Mn呈显著正相关,与N、P、K、Mg呈显著负相关.     

不同竹龄雷竹中硅及其他营养元素吸收和积累特征

在浙江省临安市的雷竹主产区,分别采集不同竹龄(1～4 a)和不同器官(叶、枝、秆)的雷竹样品,分析了Si和其他营养元素含量、吸收和积累特征,以及Si和其他营养元素之间的相互关系.结果表明： 雷竹各器官中C含量的大小顺序为竹秆>竹枝>竹叶,Si、N、P、K、Ca、Mg、Al、Fe和Mn含量的大小顺序为竹叶>竹枝>竹秆.除Mn主要积累在竹叶中外,其他9种营养元素主要积累在1年生雷竹的秆中.3～4年生雷竹竹叶的Si平均含量为13.66 g · kg^-1. 雷竹属于Si积累植物.随竹龄的增加,雷竹叶中的N、P、K和Mg含量减少,C、Al和Mn含量增加.雷竹对Si的吸收主要集中在第2年(57.1%),对N和K的吸收主要集中在前两年(67.7%～93.7%),此后N和K从植株体内流出,其流失量分别占总积累量的19.1%～39.1%.雷竹中Si与Ca、Al、Mn呈显著正相关,与N、P、K、Mg呈显著负相关.     

不同抚育措施对闽西毛竹林碳密度、碳贮量与碳格局的影响

以闽西永安市Ⅰ（挖笋+劈草+施肥+灌水)、Ⅱ(挖笋+劈草+施肥)、Ⅲ（挖笋+劈草)3种不同抚育措施的毛竹林为对象,对其碳密度、碳贮量及碳格局进行了研究。结果表明：毛竹各器官碳密度变动范围为0.3947～0.5619 g·g^-1,碳密度高低排序为竹鞭＞竹秆＞竹枝＞竹蔸＞竹叶＞竹根＞鞭根;不同竹龄间竹秆、竹枝、竹叶、竹根碳密度存在极显著差异（P<0.01）,而竹蔸碳密度差异不显著（P＞0.1）;随着竹龄增加,毛竹平均碳密度有下降趋势,1～6年毛竹平均碳密度为0.4579～0.4957 g·g^-1。Ⅰ、Ⅱ和Ⅲ类毛竹林毛竹层碳贮量分别为76.74、64.30和55.91 t·hm^-2;凋落物层碳贮量分别为2.59、3.01和4.88 t·hm^-2;土壤层碳贮量分别为150.64、197.36和232.56 t·hm^-2;总碳贮量分别为227.37、261.66和288.47 t·hm^-2,其中土壤层碳贮量所占比例最高,分别为66.25%、75.43%和80.62%,毛竹层碳贮量比例分别为32.61%、23.43%和17.69%,凋落物层碳贮量比例分别为1.14%、1.15%和1.69%。毛竹生物器官中,竹秆固碳能力最强,占年总固碳量比例为56.47%～59.66%,鞭根的固碳能力最弱,占年总固碳量的比例仅为2.52%～2.83%;毛竹林年固碳量排序为Ⅰ类型＞Ⅱ类型＞Ⅲ类型。     

NaCl胁迫对玉米幼苗不同器官离子含量的影响

在实验室水培条件下,研究了NaCl胁迫下玉米幼苗不同器官中Na+、K+,Ca2+,Mg 2+和Cl-含量的变化.结果表明:玉米各个部分Na+和Cl-含量、Na+/K+和Na+/Ca2+比值均随着培养液中NaCl浓度的增加而迅速提高,Na+,K+和Cl-含量的变化幅度为根系>成熟叶叶鞘>生长叶>成熟叶叶片,玉米幼苗根系最易受外界离子浓度的影响,叶片受外界环境影响较小;各器官中Ca2+、Mg2+对盐胁迫的响应不一致,NaCl胁迫使根系中Ca2+、Mg2+含量下降,成熟叶叶鞘中Mg2+含量变化规律性不明显,而NaCl胁迫下,成熟叶叶片中Ca2+、Mg2+含量增加;玉米幼苗具有拒Na+机制,具有一定的耐盐性,它的耐盐性是通过根和成熟叶叶鞘来实现的,Na+主要贮存在根系和成熟叶叶鞘中,而向成熟叶叶片和生长叶中运输较少;成熟叶叶鞘同时还具有拒Cl-能力.     

NaCl胁迫对白刺试管苗渗透调节物质及离子含量的影响

以西伯利亚白刺试管苗为材料,通过组织培养法研究了其在0、25、50、100和200 mmol·L-1 NaCl 胁迫40 d后的生长指标、有机渗透调节物质含量和Na+、K+、Ca2+、Mg2+离子含量的变化,以探讨其耐盐性.结果表明:(1)白刺试管苗地上部干重和根干重在50 mmol·L-1 NaCl胁迫下显著高于对照,而在100和200 mmol·L-1 NaCl胁迫下均显著低于对照.(2)随NaCl胁迫浓度增加,白刺试管苗叶片可溶性糖、脯氨酸含量和细胞质膜透性均呈持续上升趋势,叶片叶绿素含量和丙二醛含量分别呈先升后降、先降后升的趋势,并在50 mmol·L-1 NaCl处理时分别达到最高值和最低值.(3)随着NaCl处理浓度增加,白刺试管苗Na+含量和根系K+含量呈增加趋势且各处理均显著高于对照,幼苗Ca2+含量和地上部K+含量却呈减少趋势,而Mg2+含量较稳定;同时其Na+/K+ 、Na+/Ca2+和Na+/Mg2+随NaCl处理浓度增加而升高.研究发现,在低盐浓度(≤50 mmol·L-1NaCl)胁迫下,白刺试管苗能积累Na+离子和有机渗透调节物质,在根系中维持较高水平的K+和Ca2+含量以及较低水平的Na+/K+和Na+/Ca2+比,以降低白刺细胞渗透势来适应盐渍环境,从而保持其较高的生长水平.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b_6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b₆f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c_i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b₆f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c₃ isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Spatio-temporal variability in the photosynthetic characteristics of Zostera tasmanica measured by PAM

Rapid light curves (RLCs), based on pulse amplitude modulated (PAM) fluorometry, were used to investigate the spatio-temporal variability in photosynthesis versus irradiance parameters (α, I_k and P_max) and the F_v/F_m ratio of the seagrass Zostera tasmanica (formerly Heterozostera tasmanica). Spatial variation was examined across scales ranging from within a leaf (cms) to across the bed (ms), using a nested analysis of covariance sampling design. Overall, significant variation was identified at all scales examined, excluding the largest scale (area). Patterns of variability differed among individual parameters; however a high percentage of the variation was consistently assigned to the covariates, age (within and between leaves) for all parameters, except P_max.     

Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc₁ complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc₁ complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 °C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Å resolution.     

Evidence of functional trimeric chlorophyll a/c2-peridinin proteins in the dinoflagellate Symbiodinium

The chlorophyll a-chlorophyll c₂-peridinin-protein (apcPC), a major light harvesting component in peridinin-containing dinoflagellates, is an integral membrane protein complex. We isolated functional acpPC from the dinoflagellate Symbiodinium. Both SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) analysis quantified the denatured subunit polypeptide molecular weight (MW) as 18 kDa. Size-exclusion chromatography (SEC) and blue native gel electrophoresis (BN-PAGE) were employed to estimate the size of native acpPC complex to be 64–66 kDa. We also performed native ESI-MS, which can volatilize and ionize active biological samples in their native states. Our result demonstrated that the native acpPC complex carried 14 to 16 positive charges, and the MW of acpPC with all the associated pigments was found to be 66.5 kDa. Based on these data and the pigment stoichiometry, we propose that the functional light harvesting state of acpPC is a trimer. Our bioinformatic analysis indicated that Symbiodinium acpPC shares high similarity to diatom fucoxanthin Chl a/c binding protein (FCP), which tends to form a trimer. Additionally, acpPC protein sequence variation was confirmed by de novo protein sequencing. Its sequence heterogeneity is also discussed in the context of Symbiodinium eco-physiological adaptations.     

异育银鲫GABAB受体1亚基cDNA部分序列的克隆及表达分析

为了确定γ-氨基丁酸B受体（gamma-aminobutyric acid B receptor,GABA_BR）基因在异育银鲫（Carassius auratus gibelio）不同组织中的表达,本实验分别对异育银鲫不同组织中GABA_BR1基因进行RT-PCR扩增,并进行了克隆和测序,在与GenBank基因库中已知GABA_BR1序列进行同源性比对的基础上采用邻接法构建系统发育树,并进一步分析其在异育银鲫不同组织内的表达水平。经克隆获得异育银鲫GABA_BR1基因CDS区序列383 bp,编码127个氨基酸。荧光定量PCR结果显示,GABA_BR1基因在异育银鲫脑、肝、肾、心、肠、鳔、鳃、肌、尾鳍、脾、卵巢、精巢组织中均有表达,且在不同组织中的表达水平由高到低依次是：脑>尾鳍>精巢>心、肠、鳔>卵巢、脾、鳃、肌>肝、肾。本研究证实了GABA_BR1基因在异育银鲫各组织中表达的广泛性,且有明显的组织特异性。     

Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc₁ complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (b_H heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b_H heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the b_H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.