Degradation of 1,4-dichlorobenzene by Alcaligenes sp. strain A175

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.     

Degradation of 1,4-dichlorobenzene by Xanthobacter flavus 14p1.

Xanthobacter flavus 14p1 was isolated from sludge of the river Mulde by selective enrichment with 1,4-dichlorobenzene as the sole source of carbon and energy. The bacterium did not use other aromatic or chloroaromatic compounds as growth substrates. During growth on 1,4-dichlorobenzene, stoichiometric amounts of chloride ions were released. Degradation products of 1,4-dichlorobenzene were identified by gas chromatography-mass spectrometry analysis. 3,6-Dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene and 3,6-dichlorocatechol were isolated from culture fluid. 2,5-Dichloromuconic acid and 2-chloromaleylacetic acid as well as the decarboxylation product 2-chloroacetoacrylic acid were identified after enzymatic conversion of 3,6-dichlorocatechol by cell extract. 1,4-Dichlorobenzene dioxygenase, dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase activity were induced in cells grown on 1,4-dichlorobenzene. The results demonstrate that 1,4-dichlorobenzene degradation is initiated by dioxygenation and that ring opening proceeds via ortho cleavage.     

Degradation of chlorobenzenes in soil slurry by a specialized organism

The microbial degradation of monochloro-, 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene in soil slurries was examined with single compounds as well as in mixtures. The indigenous soil populations brought about the degradation of monochlorobenzene when incubated at 27°C in slurries with 29% (w/w) suspended solids. In contrast, the other chlorobenzenes persisted during an incubation period of 1 month. Supplementation with buffer, mineral salts and acetate did not significantly influence the degradation. However, inoculation withPseudomonas aeruginosa strain RHO1, a monochloro- and 1,4-dichlorobenzene-degrading organism, to a titre of 1 × 10⁵ cells/g soil, led to rapid and complete degradation of 0.8 mm growth substrate within 30 h. In addition, the strain was able to degrade 1,2-dichloro- and 1,2,4-trichlorobenzene with stoichiometric release of chloride in the presence of acetate, ethanol, monochloro- or 1,4-dichlorobenzene as growth substrates. In mixtures of chlorobenzenes the co-metabolism of 1,2-dichloro- and 1,2,4-trichlorobenzene occurred until the growth substrates monochloroand 1,4-dichlorobenzene were degraded. The degradation was faster in the slurries of garden soil containing 8% organic carbon than in soil with the lower content of 2.6%.     

Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51.

Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.     

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1

Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds. 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway. All enzymes necessary to convert 1,4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1,3-dichlorobenzene degradation. Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X. flavus 14p1. Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism. Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities. As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth. Presumably a toxic compound was formed from one of the intermediates. Received: 10 June 1996 / Revision received: 23 December 1996 / Accepted: 18 January 1997     

Streptomyces sp. is a powerful biotechnological tool for the biodegradation of HCH isomers: biochemical and molecular basis

Actinobacteria are well-known degraders of toxic materials that have the ability to tolerate and remove organochloride pesticides; thus, they are used for bioremediation. The biodegradation of organochlorines by actinobacteria has been demonstrated in pure and mixed cultures with the concomitant production of metabolic intermediates including γ-pentachlorocyclohexene (γ-PCCH); 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB), or 1,4-dichlorobenzene (1,4-DCB); 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene (1,2,4-TCB), or 1,3,5-trichlorobenzene (1,3,5-TCB); 1,3-DCB; and 1,2-DCB. Chromatography coupled to mass spectrometric detection, especially GC–MS, is typically used to determine HCH-isomer metabolites. The important enzymes involved in HCH isomer degradation metabolic pathways include hexachlorocyclohexane dehydrochlorinase (LinA), haloalkane dehalogenase (LinB), and alcohol dehydrogenase (LinC). The metabolic versatility of these enzymes is known. Advances have been made in the identification of actinobacterial haloalkane dehydrogenase, which is encoded by linB. This knowledge will permit future improvements in biodegradation processes using Actinobacteria. The enzymatic and genetic characterizations of the molecular mechanisms involved in these processes have not been fully elucidated, necessitating further studies. New advances in this area suggest promising results. The scope of this paper encompasses the following: (i) the aerobic degradation pathways of hexachlorocyclohexane (HCH) isomers; (ii) the important genes and enzymes involved in the metabolic pathways of HCH isomer degradation; and (iii) the identification and quantification of intermediate metabolites through gas chromatography coupled to mass spectrometry (GC–MS).     

Degradation of 1,2-dichlorobenzene by a Pseudomonas sp.

A Pseudomonas sp. that was capable of growth on 1,2-dichlorobenzene (o-DCB) or chlorobenzene as a sole source of carbon and energy was isolated by selective enrichment from activated sludge. The initial steps involved in the degradation of o-DCB were investigated by isolation of metabolites, respirometry, and assay of enzymes in cell extracts. Extracts of o-DCB-grown cells converted radiolabeled o-DCB to 3,4-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene (o-DCB dihydrodiol). 3,4-Dichlorocatechol and o-DCB dihydrodiol accumulated in culture fluids of cells exposed to o-DCB. The results suggest that o-DCB is initially converted by a dioxygenase to a dihydrodiol, which is converted to 3,4-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,4-dichlorocatechol is by a catechol 1,2-oxygenase to form 2,3-dichloro-cis,cis-muconate. Preliminary results indicate that chloride is eliminated during subsequent lactonization of the 2,3-dichloro-cis,cis-muconate, followed by hydrolysis to form 5-chloromaleylacetic acid.     

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

Summary Three strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Bürrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a K_s value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.     

Reductive deamination as a new step in the anaerobic microbial degradation of halogenated anilines

In this paper we report the isolation and characterization of an anaerobic enrichment culture as well as of a Rhodococcus sp. strain 2 capable of degrading 3,4-dihaloanilines under nitrate reducing conditions. Using mass spectrometry several of the intermediates formed in the process of 3,4-dichloroaniline conversion were identified. Most interesting is the observation of reductive deamination and the formation of 1,2-dichlorobenzene as one of the intermediates. Using 19F NMR and fluorinated 3,4-dihaloaniline model substrates it was corroborated that reductive deamination of the anilines to give dihalobenzene intermediates represents a new initial step in the anaerobic microbial degradation of these halogenated anilines.     

好氧氯苯降解菌的分离鉴定

【目的】分离好氧氯苯降解菌,并通过研究降解特性为应用提供理论依据。【方法】利用富集培养技术分离菌株,通过形态、生理生化反应特征及16S rRNA基因序列分析鉴定菌株,测定培养液中氯苯、其它氯苯类化合物和氯离子的浓度以及菌体细胞的密度和菌体细胞粗提液中邻苯二酚双加氧酶的活性,研究菌株的降解特性。【结果】16S rRNA基因序列相似性比较表明,分离出的菌株与乙酸钙不动杆菌(Acinetobacter calcoaceticus)的相似性高达98.5%。以初始浓度为50mg/L的氯苯为唯一碳源和能源时,120h内菌株对氯苯的降解率高达98.2%,氯离子净释放量和氯苯降解量的摩尔比范围为1:1.85-1:1.39,菌体细胞粗提液中邻苯二酚1,2-双加氧酶的平均活性为0.538U/mg蛋白质。加入葡萄糖后,菌体细胞数量和氯离子浓度明显增加,但单位细胞的氯苯降解能力明显下降。在二氯苯和三氯苯共存时,菌株对氯苯的降解能力受到明显的抑制作用,但对二氯苯有一定的降解作用,降解能力大小顺序为:1,3-二氯苯1,2-二氯苯1,4-二氯苯。【结论】分离出的好氧氯苯降解菌属于Acinetobacter属菌株,该菌株对氯苯和二氯苯均具有降解作用,可能通过邻位裂环途径降解氯苯,氯苯对菌株的降解能力和邻苯二酚1,2-双加氧酶的活性具有明显的增强作用。     

Microbial degradation of 1,3-dichlorobenzene.

A gram-negative, peritrichously flagellated rod, tentatively identified as an Alcaligenes sp., was isolated from a mixture of soil and water samples by using 1,3-dichlorobenzene as the sole carbon and energy source. During growth on 1,3-dichlorobenzene, almost stoichiometric amounts of chloride were released. Simultaneous adaptation studies, as well as enzyme studies, indicated that 1,3-dichlorobenzene was metabolized via 3,5-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene to 3,5-dichlorocatechol. Subsequently, the latter product was cleaved, yielding 2,4-dichloromuconate. No initial hydrolytic step yielding 3-chlorophenol was detected in this species.     

Biodegradation of lindane using a novel yeast strain, Rhodotorula sp. VITJzN03 isolated from agricultural soil

Lindane is a notorious organochlorine pesticide due to its high toxicity, persistence in the environment and its tendency to bioaccumulate. A yeast strain isolated from sorghum cultivation field was able to use lindane as carbon and energy source under aerobic conditions. With molecular techniques, it was identified and named as Rhodotorula strain VITJzN03. The effects of nutritional and environmental factors on yeast growth and the biodegradation of lindane was investigated. The maximum production of yeast biomass along with 100 % lindane mineralization was noted at an initial lindane concentration of 600 mg l^?1 within a period of 10 days. Lindane concentration above 600 mg l^?1 inhibited the growth of yeast in liquid medium. A positive relationship was noted between the release of chloride ions and the increase of yeast biomass as well as degradation of lindane. The calculated degradation rate and half life of lindane were found to be 0.416 day^?1 and 1.66 days, respectively. The analysis of the metabolites using GC–MS identified the formation of seven intermediates including γ-pentachlorocyclohexane(γ-PCCH), 1,3,4,6-tetrachloro-1,4-cyclohexadiene(1,4-TCCHdiene), 1,2,4-trichlorobenzene (1,2,4 TCB), 1,4-dichlorobenzene (1,4 DCB), chloro-cis-1,2-dihydroxycyclohexadiene (CDCHdiene), 3-chlorocatechol (3-CC) and maleylacetate (MA) derivatives indicating that lindane degradation follows successive dechlorination and oxido-reduction. Based on the results of the present study, the possible pathway for lindane degradation by Rhodotorula sp. VITJzN03 has been proposed. To the best of our knowledge, this is the first report on lindane degradation by yeast which can serve as a potential agent for in situ bioremediation of medium to high level lindane-contaminated sites.     

Degradation of 1,2-dichlorobenzene by a Pseudomonas sp

A Pseudomonas sp. that was capable of growth on 1,2-dichlorobenzene (o-DCB) or chlorobenzene as a sole source of carbon and energy was isolated by selective enrichment from activated sludge. The initial steps involved in the degradation of o-DCB were investigated by isolation of metabolites, respirometry, and assay of enzymes in cell extracts. Extracts of o-DCB-grown cells converted radiolabeled o-DCB to 3,4-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene (o-DCB dihydrodiol). 3,4-Dichlorocatechol and o-DCB dihydrodiol accumulated in culture fluids of cells exposed to o-DCB. The results suggest that o-DCB is initially converted by a dioxygenase to a dihydrodiol, which is converted to 3,4-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,4-dichlorocatechol is by a catechol 1,2-oxygenase to form 2,3-dichloro-cis,cis-muconate. Preliminary results indicate that chloride is eliminated during subsequent lactonization of the 2,3-dichloro-cis,cis-muconate, followed by hydrolysis to form 5-chloromaleylacetic acid.     

Toxicity of chlorobenzene on Pseudomonas sp. strain RHO1, a chlorobenzene-degrading strain

Pseudomonas sp. strain RHO1 able to use chloro- and 1,4-dichlorobenzene as growth substrates was tested towards sensitivity against chlorobenzene. Concentrations of chlorobenzene higher than 3.5 mM were found to be toxic to cells independent of pregrowth with chlorobenzene or nutrient broth. Below this concentration, sensitivity towards chlorobenzene depended on the precultivation of the cells, i.e. type of growth substrate (chlorobenzene or nutrient broth) and the concentration of chlorobenzene as the growth substrate. Cells grown in continuous culture were especially sensitive with a threshold concentration of 2.5 mM chlorobenzene. In addition to chlorobenzene, metabolites also seem to function as toxic compounds. 2-Chlorophenol and 3-chlorocatechol were isolated from cell extracts. Cleavage of 3-chlorocatechol by catechol 1,2-dioxygenase seems to be the critical step in the metabolism of chlorobenzene.     

Reductive dechlorination of all trichloro- and dichlorobenzene isomers

Abstract All three isomers of trichlorobenzene were reductively dechlorinated to monochlorobenzene via dichlorobenzenes in anaerobic sediment columns. The dechlorination was specific: 1,2,3- and 1,3,5-trichlorobenzene were solely transformed to 1,3-dichlorobenzene, while 1,4-dichlorobenzene was the only product of 1,2,4-trichlorobenzene transformation. Microorganisms were responsible for the observed transformations. Since monochlorobenzene and dichlorobenzene are mineralized by bacteria in the presence of oxygen, the process of reductive dechlorination may be an important initial step to obtain complete mineralization of otherwise recalcitrant trichlorobenzenes. This is especially true for the 1,3,5-isomer, which seems to resist biodegradation in oxic environments.     

Chlorobenzene removal efficiencies and removal processes in a pilot-scale constructed wetland treating contaminated groundwater

Low-chlorinated benzenes (CBs) are widespread groundwater contaminants and often threaten to contaminate surface waters. Constructed wetlands (CWs) in river floodplains are a promising technology for protecting sensitive surface water bodies from the impact of CBs. The efficiency and seasonal variability of monochlorobenzene (MCB), 1,4-dichlorobenzene (1,4-DCB) and 1,2-dichlorobenzene (1,2-DCB) removal, the impact of planting, and gaseous MCB emissions from the filter surface were investigated over the course of 1 year in both a vegetated pilot-scale CW and an unplanted reference plot (UR). Annual mean concentration decreases of MCB and 1,4-DCB were observed; however, annual mean 1,2-DCB removal was seen only in the upper filter layer. Planting (Phragmites australis) had a statistically significant beneficial effect on removal. The CB removal efficiency in the CW generally decreased with depth, and seasonal variations of removal were evident, with less concentration decrease during summer. Load removal efficiencies of 59-65% (262-358 mg m⁻² d⁻¹) for MCB, 59-69% (4.0-5.1 mg m⁻² d⁻¹) for 1,4-DCB and 29-42% (0.6-2.1 mg m⁻² d⁻¹) for 1,2-DCB were observed in June and July. Volatilization of MCB from the filter surface accounted for 2-4% of the total amount removed. Simple cover layers of organic materials on the filter surface were suitable for MCB emission reduction. Model calculations were carried out to estimate the MCB removal potential attributable to microbial degradation, volatilisation, and plant uptake in the CW and UR. Microbial degradation was the dominating process. The observed positive impact of plants on MCB removal was caused by improved oxygen supply (due to root oxygen release into the rhizosphere and enhanced water table fluctuations), and direct plant uptake.     

Microbial degradation of 1,3-dichlorobenzene

A gram-negative, peritrichously flagellated rod, tentatively identified as an Alcaligenes sp., was isolated from a mixture of soil and water samples by using 1,3-dichlorobenzene as the sole carbon and energy source. During growth on 1,3-dichlorobenzene, almost stoichiometric amounts of chloride were released. Simultaneous adaptation studies, as well as enzyme studies, indicated that 1,3-dichlorobenzene was metabolized via 3,5-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene to 3,5-dichlorocatechol. Subsequently, the latter product was cleaved, yielding 2,4-dichloromuconate. No initial hydrolytic step yielding 3-chlorophenol was detected in this species.     

Enrichment of hexachlorobenzene and 1,3,5-trichlorobenzene transforming bacteria from sediments in Germany and Vietnam

Bacterial cultures were enriched from sediments in Germany and Vietnam reductively dechlorinating hexachlorobenzene and the highly persistent 1,3,5-trichlorobenzene to monochlorobenzene. The main products of the reductive dechlorination of hexachlorobenzene were monochlorobenzene and dichlorobenzenes (1,2-; 1,3- and 1,4-dichlorobenzene) while no trichlorobenzenes accumulated. For the reductive dechlorination of 1,3,5-trichlorobenzene with the mixed culture from Vietnam sediment, 1,3- dichlorobenzene and monochlorobenzene were produced as intermediate and final end-product, respectively. The pattern of dechlorination did not change when the cultures were repeatedly exposed to oxygen over seven transfers demonstrating oxygen tolerance of the dechlorinating bacteria. However, reductive dechlorination of 1,3,5-trichlorobenzene was inhibited by vancomycin at a concentration of 5 mg L^?1. Vancomycin delayed reductive dechlorination of hexachlorobenzene in mixed cultures by about 6 months. When repeatedly applied, vancomycin completely abolished the ability of the mixed culture to transform hexachlorobenzene. Sensitivity to vancomycin and insensitivity to brief exposure of oxygen indicates that the dechlorinating bacteria in the mixed cultures did not belong to the genus Dehalococcoides.     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

Interaction of camel lens zeta-crystallin with quinones: portrait of a substrate by fluorescence spectroscopy.

Interaction of camel lens zeta-crystallin, an NADPH:quinone oxidoreductase, with several quinone derivatives was examined by fluorescence spectroscopy and activity measurements. Fluorescence of zeta-crystallin was quenched to different levels by the different quinones:juglone (5-OH, 1,4 naphthoquinone), 1,4 naphthoquinone (1,4-NQ), and 1,2 naphthoquinone (1,2-NQ) considerably quenched the fluorescence of zeta-crystallin, where as the commonly used substrate, 9,10-phenanthrenequinone (PQ) did not induce significant quenching. Activity measurements showed only PQ served as a substrate for camel lens zeta-crystallin, while juglone, 1,4-NQ, and 1,2-NQ were inhibitors. Thus quinones that interacted with zeta-crystallin directly inhibited the enzyme, whereas the substrate had very low affinity for the enzyme in the absence of NADPH. Another substrate, dichlorophenol indophenol (DCIP), conformed to the same pattern; DCIP did not quench the fluorescence of the enzyme significantly, but served as a substrate. This pattern is consistent with an ordered mechanism of catalysis with quinone being the second substrate. All three naphthoquinones were uncompetitive inhibitors with respect to NADPH and noncompetitive with respect to PQ. These kinetics are similar to those exhibited by cysteine- and/or lysine-modifying agents. Juglone, 1,4-NQ, and 1,2-NQ interacted with and quenched the fluorescence of camel lens alpha-crystallin, but to lesser extent than that of zeta-crystallin.