高大气CO2浓度下小麦旗叶光合能量利用对氮素和光强的响应

采用开顶式气室盆栽培养小麦,设计2个大气CO₂浓度、2个光照强度和2个氮水平的组合处理,通过测定小麦叶片光合速率-胞间CO₂浓度响应曲线和叶绿素荧光参数,来测算小麦叶片光化学速率、光合电子传递速率以及叶绿体磷酸丙糖利用效率(TPU)等参数,研究施氮量和光强对高大气CO₂浓度下小麦旗叶光合能量传递与分配的影响,以阐明全球气候变化下植物光合能量分配对光合作用适应性下调的作用机制及其氮素调控。结果表明,大气CO₂浓度升高后小麦叶片的光呼吸电子传递速率(J₀)和Rubisco氧化速率(V₀)显著下降;光合电子流的光化学传递速率(J_C)、Rubisco羧化速率(V_C)和TPU则明显升高,而且施氮后变化幅度加大;小麦叶片J_C/J_F(PSⅡ反应中心总电子流速率)和TPU/V_C显著增加,经过PSⅡ反应中心的电子流更多地进入碳同化过程,表现较高的光合速率(P_n)。遮荫提高了叶片光化学速率和PSⅡ反应中心总电子流速率(J_F),这一作用在低氮叶片尤为突出,但使得J₀和V₀明显升高,并显著降低J_C/J_F,所以P_n明显下降。正常光照条件下,增施氮素可提高小麦叶片的J_F、J_C、V_C和TPU,并使高大气CO₂浓度下J₀和V₀较正常大气CO₂浓度处理显著降低,有效地提高了植物叶片对光能的利用效率;遮荫后高大气CO₂浓度下小麦叶片J_C、V_C、TPU、J_C/J_F和TPU/V_C显著高于正常大气CO₂浓度处理,而且这一变化不受氮素水平的显著调节。因此,氮素在高大气CO₂浓度下对小麦叶片光合能量利用的调节因光强而异,正常光照下可显著改善小麦叶片对光合能量的利用状况,而遮荫后这一作用减弱。     

大气CO2升高和紫外辐射相互作用对羊栖菜生理特性的影响

为了研究经济海藻羊栖菜对大气CO2浓度增加与紫外辐射(UVR)相互作用的响应,设置两个CO2浓度(380μL/L和800μL/L)以及两种辐射处理,即PAR处理(滤除UV-A、UV-B,藻体仅接受可见光,400—700nm)和PAB处理(全波长辐射280—700nm)培养海藻,探讨了羊栖菜生长、光合作用、呼吸作用、光合色素含量、可溶性糖和蛋白以及硝酸还原酶活性的变化情况。结果表明高浓度CO2显著提高羊栖菜藻体的相对生长速率,并且紫外辐射的负面效应在高CO2处理下表现不显著。高CO2降低了藻体的光合作用速率,而UVR的负面效应和生长体现为一致性,但是羊栖菜的呼吸作用没有受到环境变化的明显影响。羊栖菜的光合色素叶绿素a和类胡萝卜素在高浓度CO2处理下明显降低,而UVR没有明显影响。环境因子对羊栖菜的可溶性糖没有影响,但是在高CO2和全波长辐射处理下,藻体可溶性蛋白的含量显著增加。同时高CO2明显提高了硝酸还原酶的活性,并且仅在高浓度CO2处理下藻体中UVR对其活性有抑制作用。CO2和UVR对羊栖菜的大多数生理特性存在明显的交互作用,在未来CO2浓度进一步增加的情况下,UVR的负面效应将会得到一定程度的缓解,这样有利于羊栖菜在养殖海区获得更高的产量。     

杉木人工林不同深度土壤CO2通量

土壤CO₂通量具有明显的时间和空间变异性。土壤温度和含水量是影响土壤CO₂通量的重要因素,同时,不同深度的土壤CO₂通量对温度和含水量变化的响应差异较大,因此,研究土壤CO₂通量和影响因素随土壤深度的变化,对于准确评估土壤碳排放具有重要意义。选择福建三明杉木人工林(Cunninghamia lanceolata)作为研究对象,利用非散射红外CO₂浓度探头和Li-8100开路式土壤碳通量系统,并使用Fick扩散法计算了0-60cm深度土壤CO₂的通量,结果表明:(1)5种扩散模型计算的表层(5cm)CO₂通量与Li-8100测量结果均具有显著相关性(P<0.01),Moldrup气体扩散模型计算结果较好。(2)土壤CO₂浓度随深度的增加而升高,但60cm深度以下土壤CO₂浓度开始降低;不同深度土壤CO₂浓度的日变化均呈现单峰型;0-60cm土壤CO₂通量日通量均值变化范围为0.54-2.17μmol m^-2 s^-1;(3)指数拟合分析显示,5、10cm和60cm深度处土壤CO₂通量与温度具有显著相关性,Q₁₀值分别为1.35、2.01和4.95。不同深度土壤含水量与CO₂通量的相关性不显著。     

UV-B辐射增强和CO2浓度倍增的复合作用对番茄生长和果实品质的影响

以番茄（Lycopersicon esculeutum）为研究对象,在人工模拟8.40 kJ·m^-2的UV-B辐射和700 μmol·mol^-1的CO₂浓度复合处理下,研究了番茄的生长和果实品质变化.结果表明,UV-B辐射使番茄的株高、鲜重、干重、总叶绿素、叶绿素a、叶绿素b、光合速率、水分利用效率、可溶性蛋白、维生素c及番茄红素等降低,导致果实品质恶化;而CO₂浓度倍增作用相反.在UV-B辐射增强和CO₂浓度倍增复合作用下,番茄的上述指标与对照相比差异不明显.分析认为,CO₂倍增与UV-B辐射增强复合处理下,CO₂的正效应作用可以减轻甚至抵消UV-B辐射的负效应.     

植被类型变化对长白山森林土壤碳矿化及其温度敏感性的影响

土壤有机质是陆地生态系统最大的碳库,土壤有机质分解速率及其温度敏感性对生态系统碳循环及其碳汇功能具有重要影响。为揭示植被类型变化对森林土壤有机质分解的影响,以长白山针阔混交林的原生林和次生林为研究对象,分别将土壤在不同水分(30%、60%和90%土壤饱和含水量(SSM))和不同温度(5、10、15、20、25和30 ℃)下培养,在为期56 d的培养期内分9次测定土壤碳矿化速率。实验结果表明:植被类型、培养温度和水分对土壤碳矿化速率具有显著影响,且三者间存在显著的交互效应(P < 0.001)。次生林土壤碳矿化累积量显著高于原生林(P < 0.05),在90% SSM和温度30 ℃培养状况下分别为346.41 μgC/g和241.01 μgC/g。包含温度和水分的双因素模型可很好地拟合土壤碳矿化速率的变化,温度和水分可共同解释土壤碳矿化速率的82.7%-95.9%变异。次生林土壤碳矿化温度敏感性(Q₁₀)显著高于原生林;水分对温度敏感性的影响较复杂,次生林在60% SSM最高,而原生林在90% SSM最高。总之,原生林遭砍伐后将会加速土壤有机质的分解,从而降低土壤有机质含量;另外,根据Q₁₀值可以预测次生林土壤有机质的分解速率对全球变暖反映更明显。     

CO2和温度升高情况下白粉菌侵染对西葫芦生长特性的影响

植物病害是降低植物产量和产品品质的重要因素,但对其在气候变化情景下如何影响植物的研究鲜见报道。利用封闭式人工气候室模拟不同环境处理,探讨了大气CO₂浓度增加和温度升高情况下白粉菌(Podosphaera xanthii)侵染对西葫芦(Cucurbita pepo)生长发育的影响。结果表明,单独CO₂加富(EC)增强了西葫芦光合作用(P<0.05),促进了植株生长和果实生产;CO₂浓度和温度同时升高(ECT)也促进了光合作用(P<0.05),加速了植株器官发育,但限制了叶片叶绿素合成和叶片面积生长,最终明显降低了植株地上部分干物质积累和果实产量(P<0.05)。和对照相比,EC处理下白粉菌的生长繁殖没有明显变化,但由于西葫芦植株的抗病性有所改善,植株病情指数略有下降;而ECT处理下白粉菌的发育繁殖明显改善,P. xanthii菌落规模和产孢能力极大提高(P<0.01),植物病情指数显著加重(P<0.01),作物严重减产(P<0.01)。可见,在未来以CO₂浓度和温度升高为特征的气候变化条件下,白粉菌倾向于加重对西葫芦的侵染。这个结论对其他葫芦科白粉病的防治管理也有借鉴作用。     

苹果三维树冠的净光合速率分布模拟

构建三维树冠光合模型可模拟出叶片净光合速率(P_n)、气孔导度(G_s)和光能利用效率(LUE)在树冠内的三维分布。以17年生纺锤形"富士"苹果树(Malus domestica Borkh. cv. ‘Fuji’)为试材,通过实测确定三维树冠内叶片和辐射分布,根据不同部位叶片最大光合速率经验公式模拟叶片P_n 在三维树冠空间内分布,并据2007-2009年测定数据拟合相关模型参数。模拟表明,苹果树冠叶片P_n 和辐射的三维分布相似,在树冠上部P_n 三维分布比较平缓,然后随辐射的减少而迅速降低。高辐射条件下(PAR=1500 μmol·m^-2·s^-1),从树冠上部3 m处降到到1 m,平均相对辐射从71.18%降到8.05%,减少了89%,叶片平均P_n从15.05 μmol·m^-2·s^-1降到1.92 μmol·m^-2·s^-1,减少了87%。单位体积小室内的总净光合速率大小主要取决于叶面积密度,部分取决于P_n。G_s三维分布与P_n相似,而LUE分布与辐射相反,中下部高,上部低。根据光合机理模型、树冠内辐射和叶面积三维分布可模拟出苹果三维树冠内叶片的P_n、G_s和LUE分布,该模型参数少,可方便用于其它果树三维光合模型构建和果树整形修剪研究。     

温度与CO2浓度升高对集胞藻PCC 6803修复UV损伤的关键基因转录量的影响

通过Taqman探针绝对定量法研究了集胞藻PCC 6803在5种不同的环境条件下:(1)25℃+400μmol/mol CO₂,(2)29℃+400μmol/mol CO₂,(3)25℃+800μmol/mol CO₂,(4)29℃+800μmol/mol CO₂,(5)25℃+1200μmol/mol CO₂,其phrA/psbA1/psbA2/psbA3等UV修复基因和16S rRNA基因的转录本拷贝数的变化情况。结果表明:温度与CO₂浓度的升高可以导致集胞藻PCC 6803的psbA2/psbA3基因和16S rRNA转录本拷贝数的大幅减少,说明温室效应将有可能导致蓝藻的UV损伤修复能力与核糖体合成能力的下降;温度升高和CO₂浓度升高对psbA2/psbA3基因和16S rRNA转录本拷贝数的联合作用表现为互相抵消,说明温度升高与CO₂浓度升高的联合作用的机制较复杂,值得深入研究。     

阳光紫外辐射对褐藻羊栖菜生长和光合作用的影响

为探讨经济褐藻羊栖菜对阳光紫外辐射变化的响应,我们在全波段阳光辐射(280－700 nm),去除UV-B辐射(320－700 nm)以及光合有效辐射PAR (400－700 nm)三种辐射条件下对其进行培养,测定了其光合作用与生长的变化。羊栖菜的生长是通过每两天测量一次藻体的湿重来测定的,光合放氧是用Clark型氧电极测定的,为了测定藻体叶绿素a和紫外吸收物质的含量,从250 nm到750 nm对羊栖菜的甲醇提取液进行扫描,叶绿素a的浓度用Porra的公式计算,紫外吸收物质的计算是根据Dunlap的方法先计算紫外吸收物质和叶绿素a的比率,然后乘以每单位藻体叶绿素a的含量。结果表明,当藻体接收较多的日辐射量时有较高的相对生长速率,当滤除UVR后,较高的太阳辐射也导致了较高的光合放氧。然而太阳紫外辐射能够抑制藻体的光合放氧和生长速率,降低叶绿素a的浓度,并且这种抑制作用随着辐射水平的升高而增强。此外,阳光紫外辐射也诱导产生了一定量的紫外吸收物质,但并不足以抵抗紫外辐射对藻体的伤害作用。     

修剪节位及修剪时期对四季蜜芒枝条生长及反季节开花的综合效应

五种模型分别运用于紫茉莉的光合—光响应及CO₂响应曲线的拟合,研究其光合效率参数的变化,探讨紫茉莉光合—光响应及CO₂响应的最适模型。结果表明:(1)紫茉莉的光合—光响应及CO₂响应改进指数模型拟合R²均为0.999,拟合效果优于非直角双曲线、直角双曲线和直角双曲线修正模型。其饱和光强和最大净光合速率分别为797.299和7.879 μmolCO₂·m^-2·s^-1,饱和CO₂浓度和最大光合能力分别为1 264.447和16.783 μmol CO₂·m^-2·s^-1,均与实测值最接近;(2)五个模型拟合和预测的均方误差(MSE)、平均绝对误差(MAE),都是改进指数模型小于其他模型。改进指数模型为紫茉莉光合—光响应及CO₂响应曲线的最佳模型,实验结果可为紫茉莉的生理生态应用研究提供参考。     

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.     

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.     

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E₂ 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E₂ titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E₂ or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E₂ raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.     

Interactions between TGF-β1 and TGF-β3 and their role in medial edge epithelium cell death and palatal fusion in vitro

In recent decades, studies have shown that both TGF-β₁ and TGF-β₃ play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-β₁ and TGF-β₃ are present. Few studies have addressed the existence of interactions between TGF-β₁ and TGF-β₃, which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-β₁ and TGF-β₃ actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-β₁ or anti-TGF-β₃ antibodies and TUNEL, added rhTGF-β₁ or rhTGF-β₃ or blocked the TGF-β₁ and TGF-β₃ action at different concentrations to WT or Tgf-β₃ null mutant palate cultures, performed in situ hybridizations with Tgf-β₁ or Tgf-β₃ riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-β₁ and TGF-β₃ in the developing palate and confirm that TGF-β₃ has a more active role in MES cell death than TGF-β₁, although both are major inductors of MES disappearance. Finally, the co-localization of TGF-β₁, but not TGF-β₃, with TUNEL in the MES allows us to suggest a possible role for TGF-β₁ in MES apoptotic clearance.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

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 patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Production and preliminary characterization of a recombinant triheme cytochrome c7 from Geobacter sulfurreducens in Escherichia coli

Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c₇ from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c₇ and expressed it in Escherichiacoli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c₇ determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c₇ of known structure.