ArsH from the cyanobacterium Synechocystis sp. PCC 6803 is an efficient NADPH-dependent quinone reductase.
The cyanobacterium Synechocystis sp. PCC 6803 possesses an arsenic resistance operon that encodes, among others, an ArsH protein. ArsH is a flavin mononucleotide (FMN)-containing protein of unknown function and a member of the family of NADPH-dependent FMN reductases. The nature of its final electron acceptor and the role of ArsH in the resistance to arsenic remained to be clarified. Here we have expressed and purified Synechocystis ArsH and conducted an intensive biochemical study. We present kinetic evidence supporting a quinone reductase activity for ArsH, with a preference for quinones with hydrophobic substituents. By using steady-state activity measurements, as well as stopped-flow and laser-flash photolysis kinetic analyses, it has been possible to establish the mechanism of the process and estimate the values of the kinetic constants. Although the enzyme is able to stabilize the anionic semiquinone form of the FMN, reduction of quinones involves the hydroquinone form of the flavin cofactor, and the enzymatic reaction occurs through a ping-pong-type mechanism. ArsH is able to catalyze one-electron reactions (oxygen and cytocrome c reduction), involving the FMN semiquinone form, but with lower efficiency. In addition, arsH mutants are sensitive to the oxidizing agent menadione, suggesting that ArsH plays a role in the response to oxidative stress caused by arsenite.
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de Sevilla, cicCartuja, Américo Vespucio 49, 41092 Seville, Spain.
Manuel
M
Hervás
Luis
L
López-Maury
Pilar
P
León
Ana M
AM
Sánchez-Riego
Francisco J
FJ
Florencio
José A
JA
Navarro
D001151:Arsenic / Q000633:toxicity; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D038181:FMN Reductase / Q000737:chemistry / Q000235:genetics; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry* / Q000235:genetics; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D038181:FMN Reductase; D016660:NAD(P)H Dehydrogenase (Quinone); D001151:Arsenic