Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme–copper oxidase type

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Abstract

The investigation of respiratory N-oxide reduction as part of a biogeochemical process sustained by prokaryotes has roots over a century ago and has laid the groundwork for microbial nitric oxide (NO) biology and recognizing that NO is of bioenergetic importance as an electron acceptor in anaerobic environments. NO is an obligatory respiratory substrate of nitrate- and nitrite-denitrifying prokaryotes that release nitrous oxide or dinitrogen as products. We witness currently a broadening of the scope of NO functionality and an increase in awareness that other heme-based NO-metabolizing systems contribute to the overall capability of the prokaryotic cell to cope with NO both in anaerobic and aerobic environments, including the pathogen–host interface. NO reduction of newly recognized physiological importance is catalyzed by the pentaheme nitrite reductase, cytochrome c′, flavohemoglobin and flavorubredoxin. Respiratory NO reductases are heme–nonheme Fe proteins that can be classified either in a short-chain group, which are complexes with cytochrome c, or a long-chain group, which have a fused quinol oxidase domain. Even though NORs are not proton pumps, both reductase groups are structural homologues of heme–copper oxidases. As a unique case, the short-chain NOR of Roseobacter denitrificans acts on oxygen, based on a heme b3-CuB center. In turn, certain heme–copper oxidases have significant turnover rates with NO. NOR mechanisms have been proposed from oxidase active site chemistry. Besides being a respiratory substrate, NO is also a signaling molecule that triggers gene expression of the principal components of NO respiration by members of the Crp–Fnr superfamily of transcription regulators.

Introduction

In this article I will describe the biochemistry and genetics of respiratory NO metabolism by prokaryotes which is tied to the properties of a distinct type of NO reductase (NOR). This membrane-bound activity catalyzes the reduction of NO to nitrous oxide:2NO+2H++2e-N2O+H2O[E0(pH7.0)=+1.177V;ΔG0=-306.3kJ/mol].

The formal oxidation states of nitrogen in NO and N2O are +2 and +1, respectively. Since the reaction involves dimerization of a reactive nitrogen species to form the N,N bond, the overall reaction requires two electrons. NO is a radical (NOradical dot) and is related to the nitroxyl anion (NO) and the nitrosonium cation (NO+) by reduction and oxidation, respectively. The study of the respiratory transformation of nitrite to N2O by bacteria led to the discovery of NO as a biomolecule and its specific role in bacterial energy conservation. The function as a respiratory substrate, which NO exerts at a concentration intermediate to that of a signaling molecule or a toxic agent, is embedded in the prokaryotic process of denitrification. The earlier biochemical developments in bacterial NO respiration have been reviewed [1], [2], [3]. Although respiratory NOR of the heme–copper oxidase type is physiologically probably the most significant NO-reducing enzyme, this review will also draw attention to other bacterial NORs and show that the scope of metabolizing NO has significantly broadened. Acquisition of genomic data has accelerated tremendously and soon it will be possible to extract in silico the NO metabolome of a given prokaryote. Substantial advances have been made recently in the understanding of the active site structure of NOR and the reduction mechanism, although we are still in anticipation of the first crystal structure and a unified enzyme mechanism. The role of NO as a signaling molecule will be considered here inasmuch as it pertains to the regulation of the respiratory function. Not covered are the cytotoxic action of NO, effected at the pathogen–host interface [4], and the fungal P450 heme-thiolate NOR [5], [6]. Details of the entire nitrate denitrification process and its manifold ramifications into bacterial bioenergetics, protein chemistry and cell biology have been treated in two comprehensive articles that over time have not lost their substantiality [7], [8].

Section snippets

NOR, NOS, and more

The liberation of gaseous N-oxides by bacteria from fermenting plant material was described in the second half of the 19th century and termed denitrification by the French scientists U. Gayon and G. Dupetit [9]. This work predates by two years the discovery of bacterial dinitrogen fixation as another fundamental process of inorganic nitrogen metabolism and the biogeochemical nitrogen cycle. From the standpoint of nitrogen cycling, the N,N triple bond broken in dinitrogen fixation is

NO-reducing proteins reflect prokaryotic versatility

Respiratory NOR is not the only means by which the bacterial cell deals reductively with NO. A number of other metalloproteins also do reduce NO (Table 1). The products are ammonia or N2O, or both, depending on the protein system and exact reaction conditions. Bacteria have NO-metabolizing capabilities whether they are denitrifiers or not. Not included in Table 1 are bacterial metalloproteins which reduce NO (for example ribonucleotide reductase or bacterioferritin [40], [41]), but where this

Respiratory NORs are members of the heme–copper oxidase superfamily

Denitrifying bacteria express a respiratory NOR. NO in these bacteria is both reaction product of and substrate for an energy-conserving respiratory electron transport chain. The main enzymatic NO generators of nitrite-denitrifying bacteria are two types of respiratory nitrite reductases (reviewed in [1], [79]). The active site of the NirK protein is a type 2 copper atom, whereas in the NirS protein (synonymous with cytochrome cd1) it is heme d1. The Nir designations are derived from the

Properties of NorB

scNOR is a complex of the catalytic subunit NorB with the c-type cytochrome, NorC. The two subunits have sequence-deduced masses of about 17 kDa (NorC) and 53 kDa (NorB). Electrospray mass spectrometry matches the predicted mass of Paracoccus NorC of 17, 477 within one unit indicating absence of protein or cofactor modifications [117]. The mass obtained by SDS electrophoresis coincides only for NorC with the theoretical value, whereas NorB is a highly hydrophobic b-type cytochrome that deviates

Active site properties

The optical features of NOR indicate the presence of low-spin heme b and heme c and of a high-spin heme b, in the absorbance properties of the CO-reacted material [129]. The magnetic circular dichroism spectrum of NOR shows bands originating from a low-spin heme c (with histidine and methionine coordination) attributed to NorC, and a magnetically isolated low-spin heme b (with bis-histidine coordination) and a high-spin heme b3 magnetically coupled with nonheme Fe; the two heme b species are

Long-chain respiratory NOR, NorZ, is also a quinol oxidase

Genes for anaerobic nitrate respiration are located in the chemolithotrophic hydrogen bacterium R. eutropha both on the chromosome and on the 0.45-Mb plasmid. Since plasmid-borne denitrification genes are seldom observed ([176] and references therein), R. eutropha has been of interest for potential variations in the biochemistry and genetics of the denitrification system. This interest has been fully rewarded by the discovery of the quinol-dependent NOR. The two genomic elements of R. eutropha

Structural and functional variations among respiratory NORs

The sequence alignment of scNOR and lcNOR shows extended regions of structural similarity and positional conservation of the ligating histidine residues for the heme groups and FeB. Nevertheless, several modifications of the typical features of NOR can be observed (Table 3), which are also of interest in the evolutionary context. They may help us to understand the mechanism of the heme–copper oxidases in defining the elements which determine the reactivity of the active site.

R. denitrificans

NO as a signaling molecule

Denitrification is a facultative process, usually expressed in the absence or at a lowered tension of oxygen, when an N-oxide, for example nitrate, is present. Nitrate as a signaling molecule is sensed in pseudomonads by a two-component sensor-regulator system that switches on nitrate respiration [182]. However, nitrate itself is not the signaling molecule for the expression of NOR, but rather NO as one of the downstream reduction products of nitrate. A key observation for the nature of the

Acknowledgements

Work from my laboratory was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. I am indebted to H. Körner for bioinformatics assistance, and to my coworkers and colleagues whose names appear with mine in the references cited. C.S. Raman is thanked for communicating unpublished results on the NO sensor. Unpublished sequence information incorporated in this article was made freely available by Columbia University, The Institute for Genomic Research, The Joint

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