O2 sensitivity and H2 production activity of hydrogenases—A review

Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron–sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H₂ production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O₂ sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O₂ but proficient in H₂ production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O₂ tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O₂ sensitivity of the enzyme while it catalyzes H₂ production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H₂ production and other industrial processes, as well as our own perspective on future applications.     

Do the higher oxidation states of the photosynthetic O2-evolving system contain bound H2O?

A modified mass spectrometer was used to determine whether the higher oxidation states of the photosynthetic O2-evolving system contain substrate water that is not freely exchangeable with the external medium. Our data indicated that the higher oxidation states contain no appreciable bound, non-exchangeable H2O. This suggests that H2O oxidation takes place via a rapid, concerted, all-or-none mechanism rather than by a mechanism involving stable, partially oxidized, H2O-derived intermediates. These findings set definite constraints on possible mechanisms of O2 evolution.     

Resonance Raman studies of the peroxotitanate(IV) complexes K2(Ti(O2)(SO4)2)·5H2O and K2(Ti(O2)(C2O4)2)·3H2O

The resonance Raman spectra of K₂(Ti(O₂)(SO₄)₂)·5H₂O and K₂(Ti(O₂)(C₂O₄)₂)·3H₂O are recorded. The results are consistent with the triangular structure of the peroxotitanium unit, Ti(O₂), with C_2ν symmetry. The ν(OO), ν_s(TiO) and ν_as(TiO) are observed around 890, 610 and 535 cm⁻¹, respectively. The resonance effects are shown to be associated with the 425 nm absorption band. This band is assigned to the O₂²⁻ → Ti(IV) charge-transfer transition. The calculated force constant values for the O₂²⁻ and TiO bonds are 320 and 275 N m⁻¹, respectively.     

Mechanism of O2−- and H2O2-induced stimulation of sugar transport in mouse fibroblast BALB/3T3 cells

Xanthine/xanthine oxidase and H₂O₂ stimulated sugar transport. Application of superoxide dismutase and catalase to the cells showed an inhibitory effect on these agent-stimulated sugar transports. Addition of amiloride and 4-acetamide-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), which abolish the cytoplasmic alkalinization, inhibited the stimulation of sugar transport by xanthine/xanthine oxidase in the presence of catalase. The calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and trifluoperazine inhibited H₂O₂-stimulated sugar transport. These results suggest that O₂⁻ stimulates sugar transport in an intracellular pH-dependent manner and that H₂O₂ stimulates sugar transport in a calcium-calmodulin-dependent manner. These mechanisms may be involved in sugar-transport stimulation in mouse fibroblast BALB/3T3 cells by the tumor-promoting phorbol ester phorbol-12,13-dibutyrate and insulin, since the stimulatory effects of these agents were inhibited by scavengers of oxygen radicals.     

植物中的H2O2信号及其功能

H2O2是植物细胞的信号分子,是细胞正常代谢的产物,生物和非生物胁迫促使植物细胞产生H2O2,通过H2O2信号应答胁迫.H2O2信号调控一系列重要的植物生理生化过程,如系统获得抗性(SAR)和高度敏感抗性(HR)、细胞衰老与程序化细胞死亡(PCD)、气孔关闭、根的向地性、根的生长和不定根形成、细胞壁的发育、柱头与花粉的发育及相互关系等.Ca2+流动和可逆蛋白磷酸化作用是H2O2下游信号,通过MAPK级联作用于转录因子,最终调控基因的表达.H2O2调控多种基因的表达,包括编码抗氧化酶基因、调控程序化细胞死亡相关蛋白基因、生物与非生物胁迫应答蛋白基因等.     

Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts

Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 μM H(2) O(2) for 6 h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.     

Dexmedetomidine alleviates H2O2-induced oxidative stress and cell necroptosis through activating of α2-adrenoceptor in H9C2 cells

Molecular Biology Reports - Oxidative stress induced necroptosis is important in myocardial ischemia/reperfusion injury. Dexmedetomidine (Dex), an α2-adrenoceptor (α2-AR) agonist, has...     

Oxidative stress response of Blakeslea trispora induced by H2O2 during β-carotene biosynthesis

The cellular response of Blakeslea trispora to oxidative stress induced by H₂O₂ in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 μm of H₂O₂ into the medium after 3 days of the fermentation. The production of β-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H₂O₂, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H₂O₂.     

Catalytic effect of (H2O)n (n = 1–3) clusters on the reaction of HNO2 + HO → H2O + NO2 under tropospheric conditions

ABSTRACT

The effect of (H₂O)_n (n?=?1–3) on the HNO₂?+?HO → H₂O?+?NO₂ reaction has been investigated theoretically at the CCSD(T)/CBS//B3LYP/6-311?+?G(3df,2pd) level of theory, coupled with rate constant calculations by using variational transition state theory. Our results show that, when (H₂O)_n (n?=?1–3) was introduced into HNO₂?+?HO → H₂O?+?NO₂ reaction, the product of the reaction did not change, but the potential energy surface became quite complex, yielding two kinds of reactions, namely HNO₂···(H₂O)_n (n?=?1–3)?+?HO and HO···(H₂O)_n (n?=?1–3)?+?HNO₂. In all catalysed reactions with (H₂O)_n (n?=?1–3), the former reaction type is favourable than the latter one with its effective rate constant respectively larger by 6–1 orders of magnitude than that of latter one. Within the temperature range of 240–320?K, the relative impacts on water monomer are much more obvious than dimer and trimer. However, the effective rate constant with water is larger by 658%–17% times of magnitude, showing that the positive water effect is obvious under atmospheric conditions.     

H2O2对水稻Rubisco稳定性的影响

H2 O2 浓度低于 2 0mmol·L-1时 ,Rubisco分子稳定 ;高于 2 0mmol·L-1则Rubisco的大亚基之间发生交联 ,全酶发生聚沉。H2 O2 处理后 ,Rubisco表面巯基数目减少 ,对两种蛋白水解酶尤其是胰蛋白酶的敏感性增强 ,大亚基水解明显增加。H2 O2 处理只会增加Rubisco大亚基的水解程度 ,不会造成新的水解位点     

Uric acid salts of magnesium: crystal and molecular structures and thermal analysis of two phases of Mg(C5H3N4O3)2 · 8H2O

Two structurally different phases of a uric acid salt of magnesium, Mg(hydrogenurate)₂ · 8H₂O, have been prepared by crystallization from solution at pH = 7.5–8.0 and were investigated by x-ray crystallography, thermal analysis, and ir spectroscopy. Both phases are monoclinic, space group P2₁/c with a = 9.573(2), b = 14.627(3), c = 7.170(1) Å, β = 101.91(1)° (phase I) and a = 10.397(2), b = 14.306(3), c = 6.732(1) Å, β = 104.64(2)° (phase II). The crystal structures of both phases (R = 0.053 and 0.051, respectively) contain isolated octahedral [Mg(H₂O)₆]²⁺ cations, hydrogenurate monoanions, and two molecules of water of crystallization per formula unit. The structural formula representing these facts is [Mg(H₂O)₆] (hydrogenura-te)₂·2H₂O. The tautomeric form of the hydrogenurate molecule corresponds to the tri-keto form of uric acid deprotonated at N(3). Differences in bond length between uric acid and the hydrogenurate molecule may be described in terms of three additional resonance structures distributing the formal negative charge at N(3) within the pyrimidine (but not the imidazole) ring. Deprotonation at N(3) significantly decreases the internal C-N-C angle at N(3). Alternating pairs of medium-strong intermolecular N-HO hydrogen bonds lead to infinite chains of hydrogenurate molecules extending along the b axis of the unit cells in both phases. The main difference between the two phases lies in their stacking pattern of the hydrogenurate molecules. Infrared data confirm the hydrogen bonding characteristics resulting from the crystal structure analysis. Thermogravimetric measurements and differential scanning calorimetry data show that the dehydration of both phases occurs in two distinct steps with Mg(hydrogenurate)₂.6H₂O as an intermediate phase. The first dehydration step (−2H₂O) is a topotactic reaction with three-dimensional preservation of the main structure elements of the octahydrate in the structure of the hexahydrate.     

Important Role of Catalase in the Production of ��-carotene by Recombinant Saccharomyces cerevisiae under H2O2 Stress

The effect of H(2)O(2) supplement on cell growth and β-carotene productions in recombinant Saccharomyces cerevisiae CFW-01 and CFW-01 ctt1 deficiency in cytosolic catalase were investigated in shaking flasks. The results showed that supplement of H(2)O(2) (0.5 and 1.0 mM) can significantly stimulate the β-carotene production. However, β-carotene levels of CFW-01 ctt1Δ under 0.5 and 1 mM H(2)O(2) were 16.7 and 36.7% lower than those of CFW-01, respectively. Although lacking cytosolic catalase, no significant differences in cell growth were observed between CFW-01 ctt1Δ and CFW-01 under the same level of H(2)O(2) stress. These results suggest that β-carotene can act as an antioxidant to protect the recombinant yeast from H(2)O(2) oxidative damage in the absence of cytosolic catalase. However, catalase still plays an important role in the production of β-carotene under H(2)O(2) stress. If catalase can not timely decompose H(2)O(2), the free radicals such as OH· derived from H(2)O(2) can result in decrease of β-carotene concentration. Therefore, in the production of β-carotene by H(2)O(2) stress, not only the level of oxidative stress, but also the activities of catalase in cells should be considered.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料,利用药理学实验,结合分光光度法和激光共聚焦显微技术,探讨硫化氢(hydrogen sulfide,H2S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide,H2O2)的关系.结果表明,H2S清除剂次牛磺酸(hypotaurine,HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid,AOA)、羟胺(hydroxylamine,NH2OH)和丙酮酸钾(potasium pyruvate,C3H3KO3)+氨水(ammonia,NH3)均可不同程度抑制干旱诱导的气孔关闭;干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显,而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降;干旱胁迫能明显增加拟南芥保卫细胞中H2O2水平及叶片中H2S含量,提高D-/L-半胱氨酸脱巯基酶活性及基因表达量,而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响.清除H2O2可减弱干旱胁迫对H2S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应.研究结果表明H2S位于H2O2下游参与干旱诱导拟南芥气孔关闭的信号转导过程.     

H2O2介导的H2S产生参与干旱诱导的拟南芥气孔关闭

以野生型拟南芥(Arabidopsis thaliana)及其突变体(atrbohD、atrbohF、atrbohD/F、atl-cdes、atd-cdes)和过表达株系(OEL-CDes、OED-CDes)为材料, 利用药理学实验, 结合分光光度法和激光共聚焦显微技术, 探讨硫化氢(hydrogen sulfide, H₂S)在干旱诱导的拟南芥气孔关闭中的作用及其与过氧化氢(hydrogen peroxide, H₂O₂)的关系。结果表明, H₂S清除剂次牛磺酸(hypotaurine, HT)及合成抑制剂氨氧基乙酸(aminooxy acetic acid, AOA)、羟胺(hydroxylamine, NH₂OH)和丙酮酸钾(potasium pyruvate, C₃H₃KO₃)+氨水(ammonia, NH₃)均可不同程度抑制干旱诱导的气孔关闭; 干旱对OEL-CDes和OED-CDes植株气孔关闭的诱导作用明显, 而atl-cdes和atd-cdes叶片气孔对干旱胁迫反应的敏感性下降; 干旱胁迫能明显增加拟南芥保卫细胞中H₂O₂水平及叶片中H₂S含量, 提高D-/L-半胱氨酸脱巯基酶活性及基因表达量, 而对突变体atrbohD、atrbohF和atrbohD/F没有显著影响。清除H₂O₂可减弱干旱胁迫对H₂S含量和D-/L-半胱氨酸脱巯基酶活性的诱导效应。研究 结果表明H₂S位于H₂O₂下游参与干旱诱导拟南芥气孔关闭的信号转导过程。     

外源H2O2对水稻幼苗抗寒性的调节作用

在常温下用不同浓度的外源H2O2(0～20 mmol·L-1)预处理水稻幼苗,再进行12 h 6℃低温胁迫,根据幼苗相对含水量和质膜相对透性筛选最佳外源H2O2处理浓度,并分析最佳外源H2O2浓度下幼苗的渗透调节物质和活性氧相关指标的变化.结果表明:(1)0～8 mmol·L-1 H2O2预处理可以增加水稻幼苗的相对含水量,降低其质膜相对透性,并以4 mmol·L-1 H2O2的效果最佳.(2)低温胁迫后,与对照组相比,4 mmol·L-1外源H2O2预处理降低了水稻幼苗萎蔫程度,并使其总呼吸速率、交替途径容量都有增加,同时还抑制了丙二醛的含量,增加了可溶性糖、可溶性蛋白质和脯氨酸的含量.(3)外源H2O2预处理对水稻幼苗的内源H2O2含量以及O(-)/(·)2产生速率没有显著影响.研究发现,外源H2O2可以通过提高呼吸速率、降低脂质过氧化程度、增加碳氮代谢来有效增强水稻幼苗的抗寒性,它可能以一种独立于内源活性氧系统之外的方式发挥作用.     

H2O2对棕色固氮菌钼铁蛋白的损伤

棕色固氮菌固氮酶钼铁蛋白经Ｈ２Ｏ２作用后,钼铁蛋白的Ｍｏ和Ｆｅ原子含量,乙炔还原少在性,摩尔消光系数和α－螺旋度地匀显著降低,蛋白质肽链也许发生部分断一。本和纱中存在过氧化物酶。可避免Ｈ２Ｏ２对钼铁蛋白的损伤作用,表明Ｈ２Ｏ２对钼欠蛋白的金属原子簇和蛋白质结构均有明显的损伤作用,而过氧化物酶可保护固氮酶不受Ｈ２Ｏ２的破坏。