SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes

The cytochrome cd1 nitrite reductases are enzymes that catalyse the reduction of nitrite to nitric oxide (NO) in the bacterial energy conversion denitrification process. These enzymes contain two different redox centres: one covalently bound c-haem, which is reduced by external donors, and one peculiar d1-haem, where catalysis occurs. In the present paper, we summarize the current understanding of the reaction of nitrite reduction in the light of the most recent results on the enzyme from Pseudomonas aeruginosa and discuss the differences between enzymes from different organisms. We have evidence that release of NO from the ferrous d1-haem occurs rapidly enough to be fully compatible with the turnover, in contrast with previous hypotheses, and that the substrate nitrite is able to displace NO from the d1-haem iron. These results shed light on the mechanistic details of the activity of cd1 nitrite reductases and on the biological role of the d1-haem, whose presence in this class of enzymes has to date been unexplained.

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CHAPTER 4. Nitrite Reductase – Cytochrome cd1

Metalloenzymes in Denitrification - Metallobiology ◽

10.1039/9781782623762-00059 ◽

2016 ◽

pp. 59-90 ◽

Cited By ~ 1

Author(s):

Serena Rinaldo ◽

Giorgio Giardina ◽

Francesca Cutruzzolà

Keyword(s):

Nitrite Reductase ◽

Cytochrome Cd1

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A Mutant of Paracoccus denitrificans with Disrupted Genes Coding for Cytochrome c550 and Pseudoazurin Establishes These Two Proteins as the In Vivo Electron Donors to Cytochrome cd1 Nitrite Reductase

Journal of Bacteriology ◽

10.1128/jb.185.21.6308-6315.2003 ◽

2003 ◽

Vol 185 (21) ◽

pp. 6308-6315 ◽

Cited By ~ 41

Author(s):

Isobel V. Pearson ◽

M. Dudley Page ◽

Rob J. M. van Spanning ◽

Stuart J. Ferguson

Keyword(s):

Nitrite Reductase ◽

Paracoccus Denitrificans ◽

Wild Type ◽

Anaerobic Conditions ◽

Electron Mediator ◽

Intact Cells ◽

Cytochrome Cd1 ◽

Membrane Bound ◽

Electron Carriers

ABSTRACT In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc 1 complex to the periplasmic nitrite reductase, cytochrome cd 1. The periplasmic protein cytochrome c 550 has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c 550 and pseudoazurin are alternative electron carriers from the cytochrome bc 1 complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c 550. The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c 550 or both these proteins. We concluded that pseudoazurin and cytochrome c 550 are the alternative electron mediator proteins between the cytochrome bc 1 complex and the cytochrome cd 1-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

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Intramolecular electron transfer in cytochrome cd1 nitrite reductase from Pseudomonas stutzeri; kinetics and thermodynamics

Biophysical Chemistry ◽

10.1016/s0301-4622(02)00082-0 ◽

2002 ◽

Vol 98 (1-2) ◽

pp. 27-34 ◽

Cited By ~ 13

Author(s):

Ole Farver ◽

Peter M.H. Kroneck ◽

Walter G. Zumft ◽

Israel Pecht

Keyword(s):

Electron Transfer ◽

Nitrite Reductase ◽

Pseudomonas Stutzeri ◽

Intramolecular Electron Transfer ◽

Kinetics And Thermodynamics ◽

Cytochrome Cd1

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Cytochrome cd1 nitrite reductase of denitrification; a haem enzyme with an unusual structure and intriguing biogenesis

Journal of Inorganic Biochemistry ◽

10.1016/s0162-0134(97)89958-8 ◽

1997 ◽

Vol 67 (1-4) ◽

pp. 78

Author(s):

V. Fülöp ◽

P. Williams ◽

S. Baker ◽

N. Saunders ◽

A. Koppenhöfer ◽

...

Keyword(s):

Nitrite Reductase ◽

Unusual Structure ◽

Cytochrome Cd1

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Molecular interactions between multihaem cytochromes: probing the protein–protein interactions between pentahaem cytochromes of a nitrite reductase complex

Biochemical Society Transactions ◽

10.1042/bst0390263 ◽

2011 ◽

Vol 39 (1) ◽

pp. 263-268 ◽

Cited By ~ 6

Author(s):

Colin Lockwood ◽

Julea N. Butt ◽

Thomas A. Clarke ◽

David J. Richardson

Keyword(s):

Protein Interactions ◽

Nitrite Reductase ◽

Analytical Ultracentrifugation ◽

Gel Filtration ◽

Protein Protein Interactions ◽

Physiological Conditions ◽

Sulfate Reducing ◽

Redox Partner ◽

Electron Reduction ◽

Reducing Bacteria

The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2− to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein–protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer–dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2− or HSO3−. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

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