A cytochrome cd 1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus

Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd ₁-type nitrite reductase. It appeared to be a dimer of 60 kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d ₁. The isoelectric point of 8.6 was considerably higher than the pI determined for cd ₁ nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

Isolation,sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

By using the gene encoding the C-terminal part of thecd ₁-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd ₁-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed ₁ biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec _55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd ₁-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd ₁-type nitrite reductase, inactive due to the absence of hemed ₁. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.Abbreviations SDS sodium dodecyl sulfate - NBT nitroblue tetrazolium - PAGE polyacrylamide gel electrophoresis     

Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS,NirN and NirF

The periplasmic cytochrome cd₁ nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d₁. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d₁ into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d₁ insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d₁ biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d₁ biosynthesis and insertion of the cofactor into NirS.     

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d₁ heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd₁ NiR in which Tyr¹⁰ has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369–377) that the topologically homologous Tyr²⁵ plays a crucial role in controlling the activity of the cd₁ NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr¹⁰ with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe³⁺ of the d₁ heme is provided by different side-chains in different species.     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

Stoichiometry of nitrite reduction catalyzed by Pseudomonas aeruginosa nitrite-reductase

The stoichiometry of the reduction of nitrite catalyzed by Pseudomonas aeruginosa nitrite-reductase (cytochrome cd₁) has been shown to yield nitrous oxide as the final product. Gas chromatography experiments demonstrated that nitric oxide is also formed as a free intermediate. A sequential formation of NO and N₂O is discussed as proposed to the parallel formation of the two products.     

Cytochrome oxidase from Pseudomonas aeruginosa. III. Reduction of hydroxylamine

Pseudomonas aeruginosa cytochrome oxidase, which reduces nitrite and oxygen, is also capable of reducing hydroxylamine to ammonia.The K_m for hydroxylamine reduction is 6 · 10^?4M compared to 5 · 10^?5M for nitrite reduction. NADH, NADPH, reduced P. aeruginosa cytochrome c₅₅₁, and reduced P. aeruginosa copper protein were ineffective as electron donors for hydroxylamine reduction whereas reduced pyocyanine and methylene blue acted as electron mediators.Hydroxylamine reduction did not require the presence of Mn²⁺ of FAD and was not inhibited by prolonged dialysis versus sodium diethyldithiocarbamate. Cyanide, nitrite, and CO were very effective inhibitors.Removal of heme d and its reconstitution, as well as inhibition by CO, suggest that the reduction of hydroxylamine, like the reduction of nitrite or oxygen, proceeds via the heme d.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

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

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.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Nitrate respiration in Pseudomonas stutzeri A15 and its involvement in rice and wheat root colonization

Unlike most bacteria, the nitrogen-fixing rice-associated Pseudomonas stutzeri A15 disposes of three different nitrate reductases that enable conversion of nitrate to nitrite through three physiologically distinct processes, called nitrate assimilation, nitrate respiration and nitrate dissimilation. To study the role of nitrate respiration in rhizosphere fitness, a Pseudomonas stutzeri narG mutant was constructed and characterized by assessing its growth characteristics and whole-cell nitrate reductase activity in different oxygen tensions. Unexpectedly, the Pseudomonas stutzeri A15 narG mutant appeared to be a better root colonizer, outcompeting the wild type strain in a wheat and rice hydroponic system.     

Ancient hemes for ancient catalysts

Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their synthesis is achieved in cells via a complex biosynthetic machinery which is unlikely to be maintained, if unnecessary. Here we propose that ancient hemes, such as the d₁-heme of cd₁ nitrite reductase or the siroheme of bacterial and plant nitrite and sulphite reductases, are molecular fossils which have survived the evolutionary pressure because their role is strategic for the organism where they are found today. The peculiar NO-releasing propensity of the d₁-heme of P. aeruginosa NIR, recently shown by our group is, in our opinion, an example of this strategy. The hypothesis is that the d₁-heme structure might be a pre-requisite for the fast rate of NO dissociation from the ferrous form, a property which is crucial to enzymatic activity and cannot be achieved with a more common b-type heme.Key words: d1-heme, porphyrin, siroheme, nitrite reductase, sulphite reductase, nitric oxide, evolutionPseudomonas aeruginosa is a Gram-negative bacterium commonly found in soil and water, well known for its metabolic versatility; under anaerobic conditions it can use nitrate and nitrite to produce energy via the denitrification pathway. In natural environments, denitrification is the part of the biological nitrogen cycle in which nitrate is transformed into nitrogen gas; reduction of nitrate occurs in four stages each catalyzed by a specific metalloenzyme.^1,2 P. aeruginosa is also an opportunistic pathogen, capable of causing serious infections in several hosts, such as humans and plants^3,4; pathogenesis, NO metabolism and denitrification are strictly related.^5,6The conversion of nitrite (NO₂^-) to nitric oxide (NO) is catalyzed in denitrifying bacteria by the periplasmic nitrite reductases (NIR).⁷ In P. aeruginosa NIR is a heme-containing enzyme (cd₁NIR) which produces NO in the active site where the unique d₁-heme cofactor (Fig. 1) is bound. This peculiar heme is synthesized from iron-protoporphyrin IX and belongs to the isobacteriochlorines subgroup;¹ it is exclusively found in this type of bacterial NIR.Open in a separate windowFigure 1Chemical structure of the d₁-heme.Reduction of nitrite involves binding of this molecule to the reduced d₁-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. An high affinity for nitrite (and anions) of the ferrous d₁-heme is a peculiar feature of cd₁NIR.⁷ However since the product NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the quick release of NO. In our recent paper⁸ we have shown that NO dissociates rapidly from the reduced form of the specialized d₁-heme of P. aeruginosa cd₁NIR. This unexpected result indicates that cd₁NIR behaves differently from other hemeproteins, since the rate of NO dissociation is by far faster (more than 100-fold) than that measured for any other heme in the ferrous state.^8–11Our hypothesis is that the d₁-heme structure might be a prerequisite for the fast rate of NO dissociation from the ferrous form, a property which cannot be achieved with a standard b-type heme.A major consequence of our finding is that this property of the d₁-heme is essential to avoid quasi-irreversible binding of NO to the reduced heme, which would jeopardize the physiological function of the enzyme evolved to scavenge nitrite, the toxic product of nitrate reduction. From the bioenergetic view-point, the main energy-generating step in denitrification is nitrate reduction (with a net H⁺ traslocation of 2H⁺/2e^-); thus, although a complex electron transfer chain is often present, the major biological role of the reductive steps downstream of nitrate reduction is likely to be nitrite scavenging.² If the complex of NO with reduced cd₁NIR was very long lived it would hamper further reaction cycles thus resulting in the accumulation of nitrite which is toxic for the bacterium. In line with this interpretation, we have also shown very recently¹² that nitrite is able to displace NO from the ferrous enzyme; thus substrate availability is the key factor that controls the enzyme turnover.From the standpoint of molecular evolution it is accepted that bacterial denitrification is an ancient metabolic pathway which existed even before oxygen became abundant in the athmosphere. Several reports pointed out that the enzymes involved in aerobic respiration derive from those involved in the denitrification pathway. Primitive denitrifying bacteria (similar to the extant Paracoccus denitrificans) can be considered as a common ancestral symbiotic prototype of the eukaryotic mitochondrion. Indeed there is compelling evidence that modern eukaryotic oxidases evolved from bacterial NO-reductase once oxygen became available as a major oxidant.^13,14In microrganisms, other “ancient” metabolisms are represented by sulphite and nitrite reduction pathways, which were well suited for a prebiotic photoreducing environment.¹⁵ Also in these pathways several enzymes are heme-containing proteins in which modified hemes, such as siroheme, are used as cofactors.¹⁶ Interestingly also in plants siroheme is a relevant porphyrin group,¹⁷ being the cofactor of plant nitrite and sulphite reductases, required for the assimilation of inorganic nitrogen and sulphur from the environment.Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their biosynthesis is achieved in cells via branched pathways which are expensive in terms of energy consumption.^16–18 The single pathways are tightly regulated and often activated only “on demand” when the specific heme group is required. Therefore, parsimony suggests that a complex biosynthetic machinery is unlikely to be maintained, if unnecessary.We thus propose that these ancient hemes (such as the d₁-heme or the siroheme) are molecular fossils which have survived the evolutionary pressure because their role is strategic only for the organism where they are found today. The peculiar NO-releasing propensity of the d₁-heme of P. aeruginosa NIR shown by our group could be, in our opinion, an example of this strategy. A major challenge for the future is to unveil other uncommon features of these hemes.     

Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri

Immunogold labelling techniques on ultrathin sections of low temperature embedded cells yielded evidence for the periplasmic location of the respiratory enzymes N₂O reductase and nitrite reductase (cytochrome cd ₁) in Pseudomonas stutzeri strain ZoBell. Cell fractionation by spheroplast preparation and two-dimensional electrophoresis showed the absence of a membrane association of these enzymes. Immunocytochemical localization of N₂O reductase in a mutant strain deficient in the chromophore of N₂O reductase showed the gold label at the cell periphery, indicating that the copper chromophore processing takes place after export of this protein's apoform.     

Cytochrome cd1 Nitrite Reductase NirS Is Involved in Anaerobic Magnetite Biomineralization in Magnetospirillum gryphiswaldense and Requires NirN for Proper d1 Heme Assembly

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd₁-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d₁ heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d₁ heme for holo-cytochrome cd₁ assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.     

Nitrite reduction: a ubiquitous function from a pre‐aerobic past

In eukaryotes, small amounts of nitrite confer cytoprotection against ischemia/reperfusion‐related tissue damage in vivo, possibly via reduction to nitric oxide (NO) and inhibition of mitochondrial function. Several hemeproteins are involved in this protective mechanism, starting with deoxyhemoglobin, which is capable of reducing nitrite. In facultative aerobic bacteria, such as Pseudomonas aeruginosa, nitrite is reduced to NO by specialized heme‐containing enzymes called cd₁ nitrite reductases. The details of their catalytic mechanism are summarized below, together with a hypothesis on the biological role of the unusual d₁‐heme, which, in the reduced state, shows unique properties (very high affinity for nitrite and exceptionally fast dissociation of NO). Our results support the idea that the nitrite‐based reactions of contemporary eukaryotes are a vestige of earlier bacterial biochemical pathways. The evidence that nitrite reductase activities of enzymes with different cellular roles and biochemical features still exist today highlights the importance of nitrite in cellular homeostasis.     

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification

Summary The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd ₁) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c ₅₅₂ mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd ₁ lacking heme d ₁, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir^– mutants of another type that had low level synthesis of cytochrome cd ₁, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS ⁺ and nirD ⁺ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.     

SERR Spectroelectrochemical Study of Cytochrome cd 1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c 552 on Biocompatible Metal Electrodes

Cytochrome cd1 nitrite reductases (cd ₁NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd ₁NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd ₁NiR from Marinobacter hydrocarbonoclasticus (Mhcd ₁) depends on the presence of its physiological redox partner, cytochrome c ₅₅₂ (cyt c ₅₅₂), we show here a detailed surface enhanced resonance Raman characterization of Mhcd ₁ and cyt c ₅₅₂ attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd ₁ and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd ₁ // cyt c ₅₅₂ and Ag // SAM // cyt c ₅₅₂ // Mhcd ₁ constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c ₅₅₂ retains its native properties, while the redox potential of apparently intact Mhcd ₁ undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd ₁, reinforcing the idea that subtle and very specific interactions between Mhcd ₁ and cyt c ₅₅₂ govern efficient intermolecular electron transfer and catalytic activity of Mhcd ₁.     

Nitrosylation of c heme in cd1-nitrite reductase is enhanced during catalysis

The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d₁ heme per monomer (cd₁NiR), encoded by the nirS gene.     

Crystal Structure of Dihydro-Heme d1 Dehydrogenase NirN from Pseudomonas aeruginosa Reveals Amino Acid Residues Essential for Catalysis

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called “denitrification” and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd₁ nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d₁ as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d₁. The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d₁-binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d₁ at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417.