RESUMO
FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB-DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB-DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.
Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Heme/química , Metaloproteínas/química , Zinco/química , Sequência de Aminoácidos , Anabaena/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/química , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Heme/metabolismo , Cinética , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Oxirredução , Estresse Oxidativo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Zinco/metabolismoRESUMO
Quantification of the iron-nutritional status of phytoplankton is of great interest not only for the study of oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a marker for iron deficiency, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin have been frequently used as markers for determination of iron deficiency in phytoplankton. Using purified flavodoxin and ferredoxin from Scenedesmus vacuolatus and polyclonal antibodies against both proteins, individual ELISA tests have been developed. The assays have a linear response in the range of 30-600 ng/ml of protein.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Ferredoxinas/análise , Flavodoxina/análise , Deficiências de Ferro , Fitoplâncton/química , Anticorpos/química , Anticorpos/imunologia , Biomarcadores/análise , Biomarcadores/química , Clorófitas/química , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/imunologia , Imunodifusão/métodos , Modelos Lineares , Fitoplâncton/metabolismo , Sensibilidade e EspecificidadeRESUMO
Fur proteins are global regulators present in all prokaryotes. In Anabaena sp. PCC 7120 FurA controls iron uptake and modulates an important set of genes related primarily to photosynthesis, nitrogen metabolism and oxidative stress defense. Expression of furA is tuned by the cis-acting antisense alpha-furA RNA that is co-transcribed with the outer-membrane protein Alr1690. Disruption of the alpha-furA-alr1690 message produces the iron-deficient JAH3 mutant that lacks Alr1690 and shows enhanced expression of FurA. JAH3 cells present severe structural disorders related to the number, organization and density of photosynthetic membranes. Quantitative analysis of the fluorescence induction shows that the mutation affects the J-I and I-P phases and causes important alterations in the photosynthetic apparatus, leading to lower photosynthetic performance indexes. These results reveal that expression of the alpha-furA-alr1690 message is required for maintenance of a proper thylakoid arrangement, efficient regulation of iron uptake and optimal yield of the photosynthetic machinery.