DNA buoyant density shifts during 15N-DNA stable isotope probing

DNA-based stable isotope probing (SIP) is a novel technique for the identification of organisms actively assimilating isotopically labeled compounds. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for these shortcomings. Changes in DNA buoyant density (BD) resulting from ¹⁵N incorporation were determined using cultures of disparate GC content (Escherichia coli and Micrococcus luteus). Incorporation of ¹⁵N into DNA increased BD by 0.015±0.002 g mL⁻¹ for E. coli and 0.013±0.002 g mL⁻¹ for M. luteus. The DNA BD shift was greatly increased (0.045 g mL⁻¹) when dual isotope (¹³C plus ¹⁵N) labeling was employed. Despite the limited DNA BD shift following ¹⁵N enrichment, we found the use of gradient fractionation, followed by a comparison of T-RFLP profiles from fractions of labeled and control treatments, facilitated detection of enrichment in DNA samples from either cultures or soil.     

东北森林两种典型阔叶树种子和幼苗对15N同位素的富集响应

以东北森林两种典型的阔叶树种风力传播种子——花曲柳和色木槭种子为研究对象,通过室内¹⁵N尿素浸泡试验和温室盆栽试验,设置4个浓度(0、0.05、0.1和0.2 g·L^-1)、3个浸泡时间(4、8和12 d)与4个叶期(2、4、6和8叶)处理,研究种子浸泡浓度、浸泡时间和幼苗叶期对种子和幼苗¹⁵N富集的影响.结果表明: 浸泡浓度和浸泡时间对两树种种子δ¹⁵N值均有显著的正反馈作用,高浓度和长时间(0.2 g·L^-1+12 d)更有利于种子¹⁵N总量的富集,花曲柳和色木槭种子¹⁵N同位素最大富集倍数的浸泡浓度和浸泡时间组合分别为0.1 g·L^-1+(4、8 d)和0.05 g·L^-1+(4、8 d);δ¹⁵N值稀释率随幼苗株高的增加先急剧减少(2~6叶)后趋于稳定,幼苗从8叶开始叶片¹⁵N总量降低,表明6叶幼苗更适合追踪幼苗的来源;幼苗叶片δ¹⁵N值与种子浸泡浓度、浸泡时间和种子的δ¹⁵N值呈显著正相关.花曲柳和色木槭种子及幼苗均能成功富集到¹⁵N信号,采用0.1 g·L^-1+8 d+6叶组合最适合追踪花曲柳和色木槭种子和幼苗.     

东北森林两种典型阔叶树种子和幼苗对15N同位素的富集响应

以东北森林两种典型的阔叶树种风力传播种子——花曲柳和色木槭种子为研究对象,通过室内¹⁵N尿素浸泡试验和温室盆栽试验,设置4个浓度(0、0.05、0.1和0.2 g·L^-1)、3个浸泡时间(4、8和12 d)与4个叶期(2、4、6和8叶)处理,研究种子浸泡浓度、浸泡时间和幼苗叶期对种子和幼苗¹⁵N富集的影响.结果表明: 浸泡浓度和浸泡时间对两树种种子δ¹⁵N值均有显著的正反馈作用,高浓度和长时间(0.2 g·L^-1+12 d)更有利于种子¹⁵N总量的富集,花曲柳和色木槭种子¹⁵N同位素最大富集倍数的浸泡浓度和浸泡时间组合分别为0.1 g·L^-1+(4、8 d)和0.05 g·L^-1+(4、8 d);δ¹⁵N值稀释率随幼苗株高的增加先急剧减少(2~6叶)后趋于稳定,幼苗从8叶开始叶片¹⁵N总量降低,表明6叶幼苗更适合追踪幼苗的来源;幼苗叶片δ¹⁵N值与种子浸泡浓度、浸泡时间和种子的δ¹⁵N值呈显著正相关.花曲柳和色木槭种子及幼苗均能成功富集到¹⁵N信号,采用0.1 g·L^-1+8 d+6叶组合最适合追踪花曲柳和色木槭种子和幼苗.     

草甸草原土壤与植物系统自然丰度氮稳定同位素的年际变化及其指示作用

氮是陆地生态系统生产力的首要限制性养分,利用自然丰度δ¹⁵N(¹⁵N/¹⁴N)可以有效指示生态系统氮循环过程。本试验研究了内蒙古草甸草原土壤与植物系统自然丰度δ¹⁵N、土壤净氮矿化潜势的年际变化。结果表明: 2017—2020年,土壤NO₃^--N含量(9.83～14.79 mg·kg^-1)均显著高于NH₄⁺-N含量(3.92～5.00 mg·kg^-1);土壤NH₄⁺的δ¹⁵N值(13.3‰～18.3‰)显著高于NO₃^-的δ¹⁵N值(3.76‰～6.14‰),土壤NO₃^-的δ¹⁵N值与土壤NO₃^-含量呈显著负相关;干旱年NH₄⁺的δ¹⁵N值相对较高,降水较高或较低年NO₃^-的δ¹⁵N值显著降低。干旱年土壤净氮矿化速率、净氨化速率显著高于湿润年,而土壤硝化速率与年降水量无显著相关性。植物δ¹⁵N值与土壤δ¹⁵N值无显著相关性,但与植物N含量呈显著负相关;豆科植物与非豆科植物δ¹⁵N值、N含量均呈显著正相关,在一定程度上表明豆科植物对非豆科植物的N吸收具有促进作用。研究结果可为草原土壤-植物系统氮循环过程及其对降水变化的响应提供数据支撑。     

Effects of 15N application frequency on nitrogen uptake efficiency in citrus trees

Two irrigation systems were used to compare nitrogen uptake efficiency in citrus trees and to evaluate the NO₃⁻ runoff in «Navelina» orange trees [Citrus sinensis (L.) Osbeck] on Carrizo citrange rootstock (Citrus sinensis × Poncirus trifoliata Raf.). These were fertilized with 125 g N as labelled K¹⁵NO₃ and grown outdoors in containers filled with a sand-loamy soil. Two groups of 3 trees received this N dose either in five equally split applications by a flooding irrigation system or in 66 applications by drip. Trees were harvested at the end of the vegetative cycle (December) and the isotopic ratios of ¹⁵N/¹⁴N were measured in the soil-plant system. The N uptake efficiency of the whole tree was higher with drip irrigation (75 percnt;) than with flooding system (64 percnt;). In the 0-90 cm soil profile, the N immobilized in the organic fraction was similar for both irrigation methods (around 13 percnt;), whereas the N retained as NO₃⁻ was 1 percnt; of the N applied under drip and 10 percnt; under flooding. In the last case, most of NO₃⁻ remained under root system and it could be lost to leaching either by heavy rainfalls or excessive water applications. These results showed that a drip irrigation system was more efficient for improving water use and N uptake from fertilizer, in addition to potentially reduced leaching losses.     

较长时间内15N标记的氨态氮和硝态氮在小嵩草草甸中的运移规律

运用15N稳定性同位素技术,对15N标记的硝酸盐和铵盐在输入小嵩草(Kobresia pygaea C.B.Clarke)草甸11～13个月后的运移规律进行了研究.在经历11～13个月后,进入无机氮库中的15N很少,一般不超过所输入氮素的l%,而较多的1 5N为土壤有机质、土壤微生物和植物所固持.NO3--15N和NH4 -1 5N在小嵩草草甸中的运移规律差异很大.在11、12和13个月后,NO3--15N的总恢复率分别为92.83%、92.64%和79.96%;而NH4 -15N的则分别为49.6%、63.33%和66.22%.两者的差异在土壤有机质、土壤微生物和植物等库之间的分配中更加明显.输入NO3--15N时在11、12个月后植物所固持的15N最多,而土壤微生物和土壤有机质所固持的15N比较接近,而在13个月后,土壤有机质和植物所固持的15N接近,而土壤微生物所固持的15N下降许多;当输入NH4 -15N,土壤有机质所固持的1 5N比植物和土壤微生物所固持的都多,而且植物所固持的15N比较稳定,而土壤微生物所固持的15N则有较大变化.这表明在较长的时间内嵩草草甸对NO3-和NH4 的固持能力是不一样的.     

Use of 15N stable isotope to quantify nitrogen transfer between mycorrhizal plants

Aims Mycorrhizas (fungal roots) play vital roles in plant nutrient acquisition, performance and productivity in terrestrial ecosystems. Arbuscular mycorrhizas (AM) and ectomycorrhizas (EM) are mostly important since soil nutrients, including NH₄⁺, NO₃^? and phosphorus, are translocated from mycorrhizal fungi to plants. Individual species, genera and even families of plants could be interconnected by mycorrhizal mycelia to form common mycorrhizal networks (CMNs). The function of CMNs is to provide pathways for movement or transfer of nutrients from one plant to another. In the past four decades, both ¹⁵N external labeling or enrichment (usually expressed as atom%) and ¹⁵N naturally occurring abundance (δ¹⁵N, ‰) techniques have been employed to trace the direction and magnitude of N transfer between plants, with their own advantages and limitations.     

铵盐和硝酸盐稳定同位素丰度测定方法及其应用案例

综述了过去几十年来铵盐和硝酸盐稳定同位素丰度测定方法的历史发展变化,分析了各种方法的优缺点,并对新方法作了介绍和推荐.目前铵盐稳定同位素丰度的最新测定方法为次溴酸盐氧化结合羟胺还原法,硝酸盐氮氧同位素丰度主流的测定方法为反硝化细菌法和镉粉叠氮酸还原化学法.这些方法的主要共同特点是以N₂O为分析物,分析精度高,对样品的含氮量需求小,一般只需要10～60 nmol N,适用于低浓度样品.新方法的建立对于国内外开展氮素循环研究将起到极大的推动作用.     

Synthesis and characterization of trisodium salt of bis(thiosalicylato)aurate(I): Na3[Au(TSA)2]·5H2O

Sodium salt of a water-soluble, anionic, and monomeric 1:2 complex of Au(I) with a dianion of thiosalicylic acid TSA²⁻(Hin2TSA) = o-HS(C₆H₄)COOH) was first prepared and isolated as colorless needle crystals through a stoichiometric reaction of NaAuCl₄:H₂TSA:NaOH = 1:4:8 molar ratio in aqueous/EtOH solution. In this reaction, TSA²⁻ ligand has played a role of a reducing agent for the starting Au(III) ion and also of donor ligands coordinating to the reduced Au(I). This compound was characterized by complete elemental analyses, TG/DTA, FT-IR, 2D-NMR (¹H-¹H COSY, ¹H-¹³C HMBC, and ¹H-¹³C HMQC) spectroscopy, and the molmass measurement based on the cryoscopic method. It was shown that this complex was a monomeric species of Au(I) with a formula of Na₃[Au(TSA)₂]·5H₂O in the solid state, but not a polymeric species even in aqueous solution. A full assignment of seven carbon and four proton resonances in the coordinated TSA²⁻ ligand was achieved by the 2D ¹H-¹³C HMBC NMR technique.     

外源输入氮素在藓类生物土壤结皮中各氮组分的分配特征与归趋途径

针对黄土高原典型藓类生物结皮,以无结皮裸地为对照,通过野外原位同位素标记试验,在标记后1~30 d连续取样测定,示踪外源添加¹⁵N在生物结皮中各氮组分的分配特征,并分析¹⁵N在土壤-微生物-藓植株中的归趋途径,对比揭示生物结皮对土壤氮循环的影响。结果表明: 1)生物结皮的全氮、微生物生物量氮、可溶性有机氮中¹⁵N含量分别平均比裸地高2.9、17.5、9.0倍,且藓植株固定的¹⁵N含量高达4.73 mg·kg^-1。2)生物结皮的¹⁵N残留率平均为13.0%,其氮固持能力是裸地(3.3%)的4.0倍;生物结皮中各组分¹⁵N占全氮的比率依次为微生物生物量氮(54.3%)>藓植株氮(22.5%)>可溶性有机氮(6.2%),而裸地则为微生物生物量氮(11.5%)>可溶性有机氮(2.6%),显示生物结皮中微生物和藓植株的氮固持能力合计比裸地高65.3%。3)生物结皮中微生物生物量¹⁵N的转移量和库容量分别比裸地高17.2和20.5倍,但其周转率为每月5.8次,低于裸地的每月7.2次,其周转期是裸地的1.2倍。综上,与无结皮裸地相比,生物结皮具有更高的氮固持能力,同时能够改变土壤各氮组分的分配率,因此,在干旱生态系统土壤氮循环过程中具有重要作用。     

The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition

A novel bloom of Alexandrium minutum occurred in an inner basin of the Cape Town harbour from November 2003 to February 2004. Cellular concentrations reached a maximum of 1.4 × 10⁸ cells l⁻¹ during the mid-December period with corresponding chlorophyll a concentrations of 243 mg m⁻³. Primary productivity measurements conducted during the latter part of the bloom revealed a maximum assimilation number of 11.17 mg C mg Chl a⁻¹ h⁻¹ during the middle of the day. Productivity during this post-peak period was sustained largely by the reduced nitrogen species NH₄ and urea (96%) as measured using ¹⁵N tracer techniques. The large subunit ribosomal DNA sequence of A. minutum isolates from Cape Town harbour was identical to conspecifics collected in Western Europe and in Australia. The composition of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was limited to gonyautoxins (GTX1-GTX4). This profile combined with evidence of a low toxin cell quota (1.5 fmol GTX cell⁻¹) supports a close association of this taxon with other members of the A. minutum species complex, particularly from Europe. Toxin analysis from black mussels collected during this bloom indicated that the accumulated PSP toxins originated from A. minutum and not from Alexandrium catenella as is most often the case along the South African coast.     

供钾水平对苹果砧木M9T337幼苗生长、光合特性与15N、13C吸收利用的影响

以苹果M9T337幼苗为试材进行水培试验,采用¹⁵N和¹³C同位素示踪技术,研究不同供钾水平(0、3、6、9、12 mmol·L^-1,分别以K₀、K₁、K₂、K₃、K₄表示)对M9T337幼苗生长、光合特性与¹⁵N、¹³C吸收利用的影响.结果表明: K₂处理M9T337幼苗各器官干质量、根系长度、根系表面积、根尖数和根系活力均显著高于其他处理.叶片净光合速率(P_n)随着供钾水平的升高先上升后下降,在K₂处理时达到最高值,为15.5 μmol CO₂·m^-2·s^-1.处理30 d后,硝酸还原酶(NR)和碳代谢酶活性均以K₂处理最高,K₀处理最低.随着供钾水平的提高,各处理幼苗的¹³C积累量呈先升高后降低的趋势,且在K₂处理时各器官¹³C分配率最均衡.各处理间¹⁵N吸收量和利用率差异显著,K₂处理下幼苗的¹⁵N吸收量和利用率最高,分别为16.11 mg和17.9％,是K₀处理的3.0倍.因此,钾素供应过低或过高均抑制幼苗根系生长和叶片光合作用,不利于植株碳氮吸收,而适宜的钾素供应水平可以提高根系活力和净光合速率,增强硝酸还原酶(NR)和碳代谢酶活性,从而促进碳氮代谢.     

Growth and nitrogen status of cotton (Gossypium hirsutum L.) under salt stress revealed using 15N-labeled fertilizer

利用¹⁵N同位素揭示了盐胁迫下棉花氮素的吸收与生长状况     

钨酸钠对苹果幼苗15N吸收利用、13C积累及果实品质的影响

分别以1年生苹果砧木M9T337幼苗和5年生烟富3/SH6/平邑甜茶为试材,开展盆栽和田间试验,并结合¹⁵N、¹³C同位素示踪技术,研究不同浓度(0、0.5、1、1.5 mmol·L^-1,分别用CK、T₁、T₂和T₃表示)硝酸还原酶(NR)抑制剂钨酸钠对苹果幼苗¹⁵N吸收利用、¹³C积累和成熟期果实品质的影响。结果表明: 盆栽试验中,喷施0.5~1.0 mmol·L^-1钨酸钠可显著抑制幼苗地上部生长,但对根系生长影响不显著;当钨酸钠浓度达到1.5 mmol·L^-1时可显著抑制根系生长。同一时期各处理叶片NR活性与钨酸钠浓度呈负相关,均表现为CK>T₁>T₂>T₃。随处理时间的延长,叶片硝态氮含量总体表现为先升高后降低的趋势,同一时期各处理硝态氮含量与钨酸钠浓度呈正相关,均表现为T₃>T₂>T₁>CK。喷施钨酸钠可不同程度地降低幼苗各器官¹⁵N吸收量和¹⁵N利用率,且钨酸钠浓度越高,抑制幼苗氮素吸收和利用的效果越显著。随钨酸钠浓度的提高,地上部¹³C积累量呈先升高后降低的趋势,在T₂处理时达到最高;幼苗整株¹³C积累量呈相似的规律。田间试验结果表明,喷施钨酸钠可降低成熟期叶片和果实的氮含量,果皮花青苷含量、果实可溶性固形物、可溶性糖含量和糖酸比均不同程度提高,其中T₂处理的效果最好。综上,T₂处理(1.0 mmol·L^-1钨酸钠)可有效抑制幼苗地上部生长,降低¹⁵N吸收利用,提高¹³C积累,有利于果实品质的提高。     

库尔勒香梨春季施用15N-尿素的吸收、分配和利用特性

以6年生库尔勒香梨为试材,在春季香梨萌芽前施用¹⁵N尿素,研究香梨施用¹⁵N尿素的吸收、分配和利用特性.结果表明:不同生育期香梨吸收的¹⁵N在各器官的分配率存在显著差异,盛花期¹⁵N优先分配在根中,其Ndff(从肥料中吸收的¹⁵N量对该器官全氮量的贡献率)最高,新稍次之;新梢旺长期和果实膨大期根部吸收的¹⁵N优先向新生器官(叶和新稍)运转,根部¹⁵N的分配率不断下降;果实成熟期果实成为新的分配中心,其Ndff最高,果实累积的¹⁵N量占香梨树体总的¹⁵N吸收量的19.8%.香梨树体对土施¹⁵N-尿素肥料的当季利用率随生育期的推进而不断提高,到果实成熟期达到最大值(18.5%).     

大豆叶面施氮量及氮素利用率的~(15)N标记

为研究大豆(Glycine max)叶面适宜施氮量及叶面氮素吸收与利用的规律,以黑龙江省三江平原大豆主栽品种合农64为实验材料,采用15N标记示踪法在大豆需氮关键时期R5期进行叶面施氮,分析大豆组织器官标记氮素的积累量及回收率。结果表明:在4.5 kg·hm–2(N3)施氮条件下,大豆组织器官干物质量及氮素积累量显著高于其它处理,其中籽粒干物质平均重22.7 g,总干物质平均重73.2 g,分别比不施氮处理(N0)高17.92%和16.38%;籽粒氮素积累量平均为134.4mg·plant–1,比不施氮处理(N0)高13.13%,说明4.5 kg·hm–2(N3)施氮条件是合农64在R5期的最适叶面施氮量。在不同施氮条件下,各组织器官标记氮积累量随着施氮量的增加呈先增加后降低的趋势,籽粒标记氮积累量在4.5 kg·hm–2(N3)施氮条件下最高,为9.96 mg·plant–1。这一结果同样说明了4.5 kg·hm–2(N3)是合农64在R5期的最适叶面施氮量,同时明确了叶面氮素是籽粒氮素积累增加的主要原因。在同一施氮水平下,各组织器官标记15N积累量顺序为籽粒茎叶荚皮叶柄根,且各器官间差异显著,说明在R5期叶面施氮籽粒积累的叶面氮素最多。从15N标记在各组织器官的贡献率来看,在3.5 kg·hm–2(N1)施氮条件下,籽粒氮素贡献率与植株氮素回收率最高,说明在叶面施氮量较小的条件下,氮素更容易被籽粒吸收利用,但净积累量却低于最适施氮量处理(N3)。在3.5 kg·hm–2(N1)施氮条件下,植株氮素回收率高于最适施氮量处理(N3)。     

库尔勒香梨春季施用15N-尿素的吸收、分配和利用特性

以6年生库尔勒香梨为试材,在春季香梨萌芽前施用¹⁵N尿素,研究香梨施用¹⁵N尿素的吸收、分配和利用特性.结果表明:不同生育期香梨吸收的¹⁵N在各器官的分配率存在显著差异,盛花期¹⁵N优先分配在根中,其Ndff(从肥料中吸收的¹⁵N量对该器官全氮量的贡献率)最高,新稍次之;新梢旺长期和果实膨大期根部吸收的¹⁵N优先向新生器官(叶和新稍)运转,根部¹⁵N的分配率不断下降;果实成熟期果实成为新的分配中心,其Ndff最高,果实累积的¹⁵N量占香梨树体总的¹⁵N吸收量的19.8%.香梨树体对土施¹⁵N-尿素肥料的当季利用率随生育期的推进而不断提高,到果实成熟期达到最大值(18.5%).     

基于多组织稳定同位素比值的热带大西洋4种鲨鱼营养生态位分化

不同代谢速率组织间的碳、氮稳定同位素比值(δ¹³C和δ¹⁵N)可以反映生物不同时间尺度的摄食信息,对探讨物种间摄食、栖息地利用和营养生态位的变化具有重要的指示作用。本研究以在大西洋热带海域兼捕的大青鲨、长鳍鲭鲨、拟锥齿鲨和尖吻鲭鲨为对象,通过测定其肌肉、肝脏和血液的δ¹³C和δ¹⁵N值,探讨4种鲨鱼营养生态位分化。结果表明: 与长鳍鲭鲨相比,尖吻鲭鲨、拟锥齿鲨和大青鲨的δ¹⁵N值相似且相对较高;大青鲨与其他鲨鱼存在摄食隔离,表现出独特的营养生态位;尖吻鲭鲨营养生态位宽幅最大,摄食食物种类和(或)栖息环境类型更多样化,其与拟锥齿鲨的营养生态位重叠度最高,说明两种鲨鱼具有潜在的资源竞争关系。尖吻鲭鲨、拟锥齿鲨和大青鲨组织间的δ¹³C、δ¹⁵N差值与其叉长均无显著相关关系,说明3种鲨鱼近期内无明显摄食变化;而长鳍鲭鲨的肝脏、血液和肌肉组织的δ¹⁵N差值与叉长显著相关,说明长鳍鲭鲨在短期内存在摄食变化。肝脏和血液的δ¹³C、δ¹⁵N值的相似性反映了两种组织整合摄食时间周期相近,其较高的代谢速率可以反映相对短时间周期的摄食信息。     

硝酸盐供应水平对平邑甜茶幼苗生长、光合特性与15N吸收、利用的影响

以霍格兰营养液为培养基质,采用¹⁵N同位素示踪技术,研究不同浓度¹⁵NO₃^--N (0、2.5、5、10和20 mmol·L^-1,分别以N₀、N₁、N₂、N₃和N₄表示)对平邑甜茶幼苗生长、光合作用、¹⁵N吸收、利用及分配的影响.结果表明:与其他处理相比,N₂处理幼苗叶绿素含量、叶面积及各器官干质量最大.叶片净光合速率(P_n)随¹⁵NO₃^--N浓度的增加显著增大,但¹⁵NO₃^--N浓度超过N₂处理后P_n略有下降.处理20 d时,N₂处理幼苗根系活力最大,根系长度、根系总表面积和根尖数也显著高于其他处理.各处理间¹⁵N分配率差异显著,N₂处理幼苗各器官间¹⁵N分配率最均衡,¹⁵N利用率也较高;随¹⁵NO₃^--N浓度增加,各处理幼苗全氮量和¹⁵N吸收量呈先升高后降低的趋势,且在N₂处理时最大,分别为103.77和21.57 mg.处理12 d后,叶片硝酸还原酶(NR)活性以N₂处理最高,N₄处理最低,至第16天时,N₄处理较N₂处理降低了84.9%.因此,¹⁵NO₃^--N供应过低抑制幼苗光合作用及氮素吸收,¹⁵NO₃^--N供应过高则抑制幼苗体内硝态氮同化及根系生长,均不利于苹果幼苗生长及氮素营养吸收利用,适量供氮有利于苹果幼苗的生长、光合作用的提高,以及氮素的吸收、利用和分配.     

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn²⁺, Fe³⁺ or Fe²⁺ are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn²⁺, Fe³⁺ and ¹H in the presence of Fe²⁺ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe²⁺SOD and Fe²⁺(Mn)SOD by ¹⁵N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe²⁺ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E_m in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH⁻ coordinated to the metal ion.