Nitrato Reductasa Complejo De Escherichia Coli K-12: Participación De Formato Deshidrogenasa Específica y Citocromo b, Componentes De Reducción De Nitrato

JOURNAL OF BACTERIOLOGY, Sept. 1969, p. 720-729 Copyright © 1969 American Society for Microbiology

Vol. 99, No. 3 Printed in U.S.A.

Nitrate Reductase Complex of Escherichia coli K-12: Participation of Specific Formate Dehydrogenase and Cytochrome b, Components in Nitrate Reduction
JOSE RUIZ-HERRERA' AND J. A. DEMOSS Department ofBiology, University of California, San Diego, La Jolla,California 92037

Received for publication 9 June 1969

The participation of distinct formate dehydrogenases and cytochrome components in nitrate reduction by Escherichia coli was studied. The formate dehydrogenase activity present in extracts prepared from nitrate-induced cells of strain HfrH was active with various electron acceptors, including methylene blue, phenazine methosulfate, and benzylviologen. Certain mutants which are unable to reduce nitrate had low or undetectable levels of formate dehydrogenase activity assayed with methylene blue or phenazine methosulfate as electron acceptor. Of nine such mutants, five produced gas when grown anaerobically without nitrate and possessed a benzyl viologen-linked formate dehydrogenase activity, suggesting that distinct formatedehydrogenases participate in the nitrate reductase and formic hydrogenlyase systems. The other four mutants formed little gas when grown anaerobically in the absence of nitrate and lacked the benzyl viologen-linked formate dehydrogenase as well as the methylene blue or phenazine methosulfate-linked activity. The cytochrome b1 present in nitrate-induced cells was distinguished by its spectral properties andits genetic control from the major cytochrome b1 components of aerobic cells and of cells grown anaerobically in the absence of nitrate. The nitrate-specific cytochrome bk was completely and rapidly reduced by 1 mm formate but was not reduced by 1 mm reduced nicotinamide adenine dinucleotide; ascorbate reduced only part of the cytochrome b1 which was reduced by formate. When nitrate was added,the formate-reduced cytochrome b1 was oxidized with biphasic kinetics, but the ascorbate-reduced cytochrome bk was oxidized with monophasic kinetics. The inhibitory effects of n-heptyl hydroxyquinoline-N-oxide on the oxidation of cytochrome b1 by nitrate provided evidence that the nitrate-specific cytochrome is composed of two components which have different redox potentials but identical spectralproperties. We conclude from these studies that nitrate reduction in E. coli is mediated by the sequential operation of a specific formate dehydrogenase, two specific cytochrome b, components, and nitrate reductase.
A number of observations indicate that nitrate reduction in Escherichia coli occurs mainly by a pathway involving formate dehydrogenase, cytochrome bl, and nitrate reductase. Amongthe various substrates which are metabolized by E. coli, formate is the most effective electron donor for the reduction of nitrate in cells grown anaerobically in the presence of nitrate (2, 23). In such cells, formate dehydrogenase, cytochrome b1, and nitrate reductase are induced to rela-

tively high levels compared to those in cells grown in the absence of nitrate (2, 17, 23). These threecomponents are present in membrane fractions (6) and have been partially purified as a unit from cell extracts (8). Finally, mutants of E. coli which are unable to produce nitrite from nitrate (NR7 mutants) lack either formate dehydrogenase or nitrate reductase or combinations of these activities and cytochrome b& (1, 16, 17, 22). The proposal that formate dehydrogenase and I Present address:Departamento de Microbiologia, Escuela cytochrome bi are specific components of the Nacional de Ciencias Biologicas, I. P. N., Carpio y Plan de Ayala, nitrate reduction system raises some important Mexico, D. F. 720

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NITRATE REDUCIASE COMPLEX OF E. COLI

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questions concerning the relationship of this pathway with other pathways of electron transport in E. coli. Formate...