Two c-Type Cytochromes, NirM and NirC, Encoded in the nir Gene Cluster of Pseudomonas aeruginosa Act as Electron Donors for Nitrite Reductase

Hasegawa, Norio; Arai, Hiroyuki; Igarashi, Yasuo

doi:10.1006/bbrc.2001.5919

Cited by 41 publications

(47 citation statements)

References 32 publications

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“…In agreement, protein interactions of NorB and NorC with NirN were detected in our study (Table 3). Furthermore, strong interactions between NorC and the c-type cytochrome NirM, which serves as an electron donor for the nitrite reductase, were measured (Table 3) (25). Additionally, an interaction of NorC with the ATP-binding protein NirQ was observed (Table 3).…”

Section: Resultsmentioning

confidence: 96%

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Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

et al. 2016

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Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa. During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO 3؊ ¡ NO 2؊ ¡ NO ¡ N 2 O ¡ N 2 . Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment. IMPORTANCEThe processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen.

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Section: Resultsmentioning

confidence: 96%

“…The P. aeruginosa enzyme is a cytochrome cd 1 carrying an electron-receiving heme c and a catalytic site with heme d 1 (3,8-dioxo-17-acrylate-porphyrindione) (22)(23)(24). The two c-type cytochromes NirM and NirC act as electron donors (25). NirN and NirF form a stable complex with the nitrite reductase NirS during enzyme maturation (26).…”

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confidence: 99%

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

et al. 2016

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“…90 Instead, two soluble cytochromes c from the nitrite reductase gene cluster were found also to be able to donate electrons to nitrite reductase at similar rates. 91 These cytochromes c are Figure 1). The figure was prepared with VMD 23 and Tachyon.…”

Section: Resultsmentioning

confidence: 99%

Coupling of Protonation, Reduction, and Conformational Change in azurin from Pseudomonas aeruginosa Investigated with Free Energy Measures of Cooperativity

Ullmann

2011

J. Phys. Chem. B

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We used free energy calculations within a continuum electrostatics model to analyze the coupling of protonation, reduction, and conformational change in azurin from Pseudomonas aeruginosa (PaAz). PaAz was characterized extensively with a variety of experimental methods. Experimentally determined pK a values and pH-dependent reduction potentials are used to validate our computational model. It is well-known from experiment that the reduction of the copper center is coupled to the protonation of at least two titratable residues (His-35 and His-83) and to the flip of the peptide bond between Pro-36 and Gly-37. Free energy measures of cooperativity are used for a detailed analysis of the coupling between protonation, reduction, and conformational change in PaAz. The reduction of the copper center, the protonation of His-35, and peptide flip are shown to be cooperative. Our results show that cooperativity free energies are useful in detecting and quantifying thermodynamic coupling between events in biomolecular systems. The protonation of His-35 and the peptide flip are found to be so tightly coupled that these events happen effectively concerted. This concerted change results in a marked alteration of the electrostatic surface potential of azurin that might affect the interaction of azurin with its binding partners.

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“…The monomeric NirN was able to bind heme d 1 in vitro and transferred the cofactor to NirS when the two proteins were mixed (12). The deletion of the nirN gene in different denitrifiers led to a mild growth phenotype, and cellfree extracts prepared from the corresponding ⌬nirN strains exhibited reduced NirS activity compared with the WT strains (9,12,16). From these observations it was deduced that NirN is not required for heme d 1 biosynthesis but, rather, might be involved in the insertion of the cofactor into NirS.…”

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confidence: 99%

“…The encoded NirN protein shares about 24% amino acid sequence identity with NirS. Moreover, NirN has been shown to be located in the periplasm and to contain a heme c cofactor (16). The monomeric NirN was able to bind heme d 1 in vitro and transferred the cofactor to NirS when the two proteins were mixed (12).…”

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confidence: 99%

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

Adamczack

Hoffmann

Papke

et al. 2014

Journal of Biological Chemistry

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Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1.

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Two c-Type Cytochromes, NirM and NirC, Encoded in the nir Gene Cluster of Pseudomonas aeruginosa Act as Electron Donors for Nitrite Reductase

Cited by 41 publications

References 32 publications

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

Coupling of Protonation, Reduction, and Conformational Change in azurin from Pseudomonas aeruginosa Investigated with Free Energy Measures of Cooperativity

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

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