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

Background: Cytochrome cd₁ nitrite reductase contains heme d₁ as an essential cofactor.

Results: P. aeruginosa lacking the NirN protein produces the heme d₁ precursor dihydro-heme d₁, which is converted by NirN to heme d₁.

Conclusion: NirN catalyzes the last step of heme d₁ biosynthesis.

Significance: The so far unknown function of NirN is revealed.

Abstract

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.

Introduction

The process of denitrification, i.e. the stepwise reduction of nitrate to molecular nitrogen, represents one important branch of the global biogeochemical nitrogen cycle. Moreover, for denitrifying organisms such as the human pathogen Pseudomonas aeruginosa, denitrification serves as a very efficient strategy for energy generation under anaerobic growth conditions (1). In the second step of the denitrification process, nitrite is reduced to nitric oxide. In P. aeruginosa, this reaction is catalyzed by the periplasmic cytochrome cd₁ nitrite reductase NirS (2). This enzyme is a homodimer of two 60-kDa subunits, each carrying a heme c and a heme d₁ cofactor (3). The non-covalently bound heme d₁ represents the catalytic site of the enzyme where the reduction of nitrite to nitric oxide takes place (4).

Heme d₁ belongs to the tetrapyrrole subfamily of isobacteriochlorins. It carries two unusual carbonyl functions on pyrrole rings A and B and an acrylate side chain on ring D that make heme d₁ unique among the naturally occurring tetrapyrroles (Fig. 1) (5, 6). The biosynthesis of heme d₁ from the common tetrapyrrole precursor uroporphyrinogen III requires six enzymatic steps (7). Most of the involved enzymes are encoded by the nir genes, which are located together in the nir operon in P. aeruginosa (nirSMCFDLGHJEN) and also in other denitrifying bacteria (Fig. 1A) (8–10). In the first step, the S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase NirE catalyzes the methylation of uroporphyrinogen III to precorrin-2 (11, 12). Next, precorrin 2 is converted to siroheme in two enzymatic steps catalyzed by a precorrin-2 dehydrogenase and a sirohydrochlorin ferrochelatase not encoded within the nir operon in P. aeruginosa. Siroheme is then decarboxylated to 12,18-didecarboxy-siroheme by the action of siroheme decarboxylase, which is composed of the four proteins NirD, NirL, NirG, and NirH (7). The penultimate step of heme d₁ biosynthesis consists of the removal of the two propionate side chains on pyrrole rings A and B, with the concomitant introduction of the carbonyl functions at these positions yielding the heme d₁ precursor dihydro-heme d₁. This transformation is believed to be catalyzed by the radical S-adenosylmethionine enzyme NirJ (7). However, this hypothesis still requires experimental proof. Similarly, the enzyme responsible for the last step of heme d₁ biosynthesis has not yet been identified. In this step, the propionate side chain on pyrrole ring D is converted to the corresponding acrylate side chain (Fig. 1B). At some stage of its biosynthesis, heme d₁, or a precursor thereof, has to be transported across the cytoplasmic membrane to be incorporated into its target enzyme NirS in the periplasm. Currently it is thought that all heme d₁ biosynthesis steps, except the last one, take place in the cytoplasm and that the final step is catalyzed in the periplasm. This hypothesis is on the basis of the finding that NirF, which has been proposed to catalyze the acrylate group formation, is a periplasmic protein (13), whereas NirE, NirDLGH, and NirJ are located in the cytoplasm.

FIGURE 1.

Heme d₁ biosynthesis. A, the nir operon from P. aeruginosa comprises the genes coding for cytochrome cd₁ nitrite reductase (nirS), electron transferring c-type cytochromes (nirM and nirC), and proteins involved in heme d₁ biosynthesis and insertion into NirS (nirF, nirD, nirL, nirG, nirH, nirJ, nirE, and nirN). The genes encoding proteins used in this study are shown as white arrows. B, the proposed last step of heme d₁ biosynthesis consists of the introduction of the double bond into the propionate side chain of pyrrole ring D of the precursor dihydro-heme d₁, yielding the acrylate side chain of heme d₁. Until now, the enzyme catalyzing this reaction was not known.

Depending on the organism, NirF is either a soluble periplasmic protein or attached to the outer face of the cytoplasmic membrane, most likely by a lipid anchor (14, 15). In addition to its periplasmic location, purified recombinant NirF from Paracoccus pantotrophus has been shown to bind heme d₁ in vitro (13). Moreover, early mutational studies with different denitrifying bacteria have shown that the deletion of the nirF gene results in bacterial strains unable to synthesize heme d₁ (8–10, 13). On the basis of all of these findings, NirF has been proposed to catalyze the formation of the acrylic double bond of heme d₁. However, so far there is no direct experimental evidence clearly attributing this enzymatic function to NirF.

Interestingly, another nir gene (nirN) is present in all denitrifying bacteria. 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 d₁ 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 cell-free extracts prepared from the corresponding ΔnirN 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 d₁ biosynthesis but, rather, might be involved in the insertion of the cofactor into NirS. In line with this proposal it has been shown that NirN from P. aeruginosa interacts with both NirS and NirF in vivo. However, in that study it was also observed by UV-visible absorption spectroscopy of periplasmic protein fractions that the cofactor content of NirS in the P. aeruginosa ΔnirN strain was different from that in the P. aeruginosa WT strain (15). Later, the same observation was reported for a ΔnirN strain of Magnetospirillum gryphiswaldense (17).

The apparently altered cofactor content of NirS in these ΔnirN strains can be rationalized by two different hypothetical scenarios: (i) NirN is involved in heme d₁ insertion into NirS, and the absence of NirN leads to an improper incorporation of the cofactor causing the observed spectral differences, or (ii) NirN is involved in the last step of heme d₁ biosynthesis (together with NirF?), and the absence of NirN leads to the accumulation of dihydro-heme d₁, which is nevertheless inserted into NirS, leading to a different form of holo NirS with different spectral properties and reduced enzyme activity. Therefore, the aim of this study was to investigate and clarify the still enigmatic function of NirN during either heme d₁ biosynthesis or insertion into NirS.

EXPERIMENTAL PROCEDURES

Chemicals

Chemicals and reagents were obtained from Sigma-Aldrich (Taufkirchen, Germany), Merck (Darmstadt, Germany), or Thermo Fisher Scientific Inc. (Schwerte, Germany). Restriction endonucleases were purchased from New England Biolabs (Frankfurt am Main, Germany). QIAquick PCR purification and gel extraction kits were purchased from Qiagen GmbH (Hilden, Germany). Q-Sepharose Fast Flow and SP-Sepharose Fast Flow were obtained from GE Healthcare. Strep-Tactin-HC resin, Desthiobiotin, and Avidin were purchased from IBA GmbH (Göttingen, Germany). All oligonucleotide primers were obtained from Metabion International AG (Martinsried, Germany).

Plasmids and Strains

The Escherichia coli strain BL21(DE3) was used as a host for protein production of recombinant semi-apo NirS (NirS_s.a.), NirN, and NirF. For the production of NirS_s.a. and NirN, E. coli BL21(DE3) was transformed with the plasmid pEC86 (provided by Dr. Linda Thöny-Meyer (18)) and either pET-22b(+)nirS (15) or pET-22b(+)StrepIInirN. For the production of recombinant NirF from Dinoroseobacter shibae, E. coli BL21(DE3) was transformed with pET-22b(+)nirF_(D.s.)PCSStrepII. The P. aeruginosa PAO1 WT strain was used for protein production of native holo NirS (NirS_WT). For protein production of native NirS containing dihydro-heme d₁ (NirS_ΔN), the P. aeruginosa PAO1 strain RM361 (nirN::tet) was used (provided by Dr. Hiroyuki Arai (9)).

Construction of Vectors

For the construction of a NirN expression vector, the nirN gene from P. aeruginosa PAO1 was PCR-amplified using the primers strepIInirN_fw (TAT ACC ATG GGC TGG AGC CAC CCG CAG TTC GAA AAA GCT AGC GGC GAA GCG CCG GGG GAA G) and nirN_rev (CG GGA TCC TCA GTG CGA GGT TC), which contained NcoI and BamHI restriction sites (underlined), respectively. The digested nirN insert was ligated into the correspondingly digested vector pET-22b(+) to generate pET-22b(+)StrepIInirN. For the construction of an expression vector for NirF from D. shibae, the plasmid pMK-RQrbspelBnirF_(D.s.)PCShis₆ was purchased from Invitrogen. Through restriction of the plasmid with NdeI and HindIII, the insert pelBnirF_(D.s.)PCShis₆ was obtained and ligated into the correspondingly digested plasmid pET-22b(+). A Strep tag coding insert was obtained by annealing the primers CTA GTA GCG CTT GGA GCC ACC CGC AGT TCG AAA AAG and GAT CCT TTT TCG AAC TGC GGG TGG CTC CAA GCG CTA. The His tag coding sequence was removed from pET-22b(+)pelBnirF_(D.s.)PCShis₆ using SpeI and BamHI restriction sites, and the Strep tag coding insert was ligated into pET-22b(+)pelBnirF_(D.s.)PCS to generate pET-22b(+)nirF_(D.s.)PCSStrepII.

Growth Conditions

E. coli BL21(DE3) carrying the plasmids pEC86 and pET-22b(+)nirS or pET-22b(+)StrepIInirN for the production of NirS_s.a. (containing the covalently bound heme c but no heme d₁) and heme c containing NirN, respectively, were grown according to Studier (19) in self-inducing ZYM5052 medium containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol for 4 h at 37 °C and, subsequently, for 26 h at 25 °C. E. coli BL21(DE3) carrying the plasmid pET-22b(+)nirF_(D.s.)PCSStrepII was grown in terrific broth (20) containing 100 μg/ml ampicillin to an optical density of 0.8 at 37 °C and, after induction with 100 μm isopropyl 1-thio-β-d-galactopyranoside, for 26 h at 17 °C. For the production of native NirS containing heme c and heme d₁ or dihydro-heme d₁, P. aeruginosa PAO1 WT or RM361, respectively, were grown under anaerobic conditions for 24 h at 37 °C in autoinducer bioassay minimal medium (according to Heydorn et al. (21)) supplied with 75 mm NaNO₃ and 7.5 μm 5-aminolevulinic acid.

Purification of Proteins

The purifications of recombinant P. aeruginosa NirS_s.a., native holo NirS_WT, and NirS_ΔN were performed according to Parr et al. (22). Recombinant NirN and NirF were purified using Strep-Tactin-HC resin according to the instructions of the manufacturer (IBA GmbH) and with 50 mm Tris-HCl (pH 7.5), 150 mm NaCl as the buffer.

Determination of Protein Concentration

Bradford reagent (Sigma-Aldrich) was used to determine protein concentrations. The reagent was used according to the instructions of the manufacturer using BSA as a standard.

Extraction of Nitrite Reductase Cofactors

Heme d₁ and dihydro-heme d₁ were extracted from purified NirS_WT and NirS_ΔN, respectively. Proteins at a concentration of 90 μm in buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl) were precipitated with 6 mm HCl. Two volumes of ethyl acetate (HPLC grade) were added, and the samples were mixed for 1 min and then centrifuged for 2 min at 13000 × g. The organic cofactor-containing phase was removed and dried in vacuo. For HR-ESI-MS analysis,³ proteins were transferred to double distilled water prior to the extraction of the cofactors, and the dried cofactors were dissolved in methanol.

UV-visible Absorption Spectroscopy

UV-visible absorption spectra of purified proteins and extracted cofactors were recorded using a V-650 spectrophotometer (Jasco, Gross-Umstadt, Germany). The reduced forms of proteins and isolated cofactors were obtained by addition of a few grains of sodium dithionite.

Resonance Raman Spectroscopy

RR spectra with a spectral resolution of 2 cm⁻¹ were recorded using a confocal Raman spectrometer (Lab Ram HR-800, Jobin Yvon) equipped with a liquid nitrogen-cooled charge-coupled device camera. The 442-nm excitation line of a He-Cd laser (Soliton) was focused onto the sample (2 μm in diameter, 1 milliwatt at the sample). Using a Linkam Cryostage THMS600 cryostat, the temperature of the sample was set to 80 K throughout the measurement. Raman shifts were calibrated to an accuracy of 0.5 cm⁻¹ using toluene as an external standard. All spectra were corrected by polynomial background subtraction.

Heme d₁ and Dihydro-Heme d₁ Binding to NirN and NirF

For binding of heme d₁ and dihydro-heme d₁ to NirN and NirF, the in vacuo dried cofactors were directly dissolved in 150 μl of buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl) containing either NirN at a concentration of 20 μm or NirF at a concentration of 38 μm. Directly after mixing, UV-visible absorption spectra of the mixtures were recorded on a V-650 spectrophotometer (Jasco).

NirN Activity Assay, Transfer of Cofactors to NirS_s.a., and Reconstitution of NirS_s.a.

The in vitro NirN activity assay was performed under aerobic or anaerobic conditions in an anaerobic chamber (Coy Laboratories, Grass Lake, MI). For the aerobic assay, dried dihydro-heme d₁ was dissolved in buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl) and transferred to a quartz cuvette (Hellma Analytics, Müllheim, Germany). NirN was subsequently added at a final concentration of 20 μm to give a final volume of 150 μl. For the anaerobic assay, the dried dihydro-heme d₁ was dissolved in degassed buffer in the anaerobic chamber, followed by the addition of 20 μm NirN under anaerobic conditions. The NirN-catalyzed reaction was monitored using a V-650 spectrophotometer (Jasco). For the reconstitution of NirS_s.a. with heme d₁ or dihydro-heme d₁ and the transfer of the NirN reaction product to NirS_s.a., the enzyme was added to the NirN assay or the cofactor solution at a final concentration of 20 μm. For the oxidation of heme d₁, sodium persulfate was added.

HR-ESI-MS (Accurate Mass Measurements) of Nitrite Reductase Cofactors

The sample solutions obtained after cofactor extraction were used for accurate mass measurements on an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany). Electrospray measurements in positive mode were performed in direct infusion mode using a custom-made microspray device mounted on a Proxeon nanospray ion source. The microspray device allows sample infusion without sheath gas through a stainless steel capillary (90-μm inner diameter) at a flow rate of ∼1 μl/min. The spray voltage was set between 2.0 and 2.3 kV. The resolution of the Orbitrap mass analyzer was set to 100,000 full width at half-maximum (FWHM) at m/z 400. Accurate mass measurements were performed using the lock mass option of the instrument control software using the cation of tetradecyl-trimethylammonium bromide (256.29988 u) as an internal mass reference, which was added to the sample solution in appropriate amounts.

RESULTS AND DISCUSSION

Purification of Native NirS from the P. aeruginosa PAO1 ΔnirN Strain RM361

To obtain more insights into the role of NirN, the first aim of this study was to purify NirS from the P. aeruginosa PAO1 ΔnirN strain RM361 and to characterize the cofactor content of this apparently different form of holo NirS. Previous attempts to purify native NirS from the P. aeruginosa PAO1 strain RM361 only resulted in the isolation of semi-apo NirS (NirS_s.a.) completely lacking heme d₁ (15). Therefore, the purification protocol was changed slightly, including the reduction of the column volume of the anion exchange chromatography column. Using the new purification procedure, NirS was purified to apparent homogeneity (Fig. 2A). The enzyme purified from strain RM361 (NirS_ΔN) exhibited a less intense green color in contrast to the green color of NirS isolated from the WT strain (NirS_WT) and the red colored semi-apo form of NirS lacking heme d₁. This observation clearly supports the idea that a different form of holo NirS was produced in the P. aeruginosa ΔnirN strain.

FIGURE 2.

Purification and characterization of NirS_WT and NirS_ΔN. A, SDS-PAGE analysis of purified native NirS_WT and NirS_ΔN and recombinant NirS_s.a. from P. aeruginosa. Lane M shows marker proteins with M_r values indicated. B and C, UV-visible absorption spectra of purified native NirS_WT and NirS_ΔN in the oxidized (as-isolated) (B) and dithionite-reduced (C) forms. Characteristic absorption bands are labeled. AU, absorption units.

UV-visible Absorption Spectroscopy of Purified NirS_ΔN and NirS_WT

To characterize the cofactor content of NirS_ΔN, UV-visible absorption spectra of the purified enzyme were recorded of the oxidized (as isolated) and dithionite-reduced forms and compared with the corresponding spectra of purified NirS_WT (Fig. 2, B and C). The absorption spectra of NirS_ΔN are clearly different from those of NirS_WT in both the oxidized and reduced forms. In fact, the spectra of the two enzymes differ, particularly in the region of the characteristic heme d₁ absorption bands. Although the spectrum of oxidized NirS_WT exhibits a typical heme d₁ peak at 642 nm, the spectrum of NirS_ΔN shows a peak at 626 nm. In the reduced form, the heme d₁ absorption band of NirS_WT is shifted to 649 nm, whereas, for NirS_ΔN, a peak shift to 631 nm is observed. These spectral differences between the purified NirS_ΔN and NirS_WT confirmed the presence of two different forms of holo NirS depending on whether the enzyme was isolated from P. aeruginosa PAO1 strain RM361 or from the WT strain. There were two possible explanations for the observed spectral differences. Either heme d₁ was not properly inserted into NirS_ΔN and the observed differences were due to a different ligation of the cofactor, or it was not heme d₁ but the precursor dihydro-heme d₁ that was present in NirS_ΔN. On the basis of the observed UV-visible absorption spectra of NirS_ΔN, we favored the second hypothesis. The conjugated system of dihydro-heme d₁ lacks one double bond compared with heme d₁. For this reason, the absorption maxima of dihydro-heme d₁ can be expected to be located at shorter wavelengths than those of heme d₁ (hypsochromic shift). Indeed, such a shift was observed for the absorption maximum of NirS_ΔN (626 nm, oxidized) compared with the heme d₁-containing NirS_WT (642 nm, oxidized).

Resonance Raman Spectroscopy of NirS_ΔN, NirS_WT and NirS_s.a.

To further characterize the unusual cofactor content of NirS_ΔN and to obtain more insights into the question of whether dihydro-heme d₁ is indeed bound in NirS_ΔN, resonance Raman (RR) spectroscopy was performed. RR spectra of purified NirS_WT, NirS_ΔN, and semi-apo NirS were measured at low temperature to reduce the strong background scattering and to increase the signal-to-noise ratio. Although, upon 413-nm excitation, the heme c bands were selectively enhanced (data not shown), 442-nm excitation led to a substantial increase of the spectral mass of heme d₁ and the putative dihydro-heme d₁ in NirS_WT and NirS_ΔN, respectively. Remaining contributions of heme c could readily be removed by subtracting the spectra of NirS_s.a. from those of NirS_WT and NirS_ΔN, using the characteristic bands of heme c as a reference (Fig. 3, C and D). Therefore, pure spectra of heme d₁ and the putative precursor were obtained in the oxidized (as-isolated) state (Fig. 3, A and B). The spectrum of ferric heme d₁ (Fig. 3A) differs substantially from that of heme c, which, because of its higher symmetry (D_4h), is dominated by the totally symmetric A_1g modes (Fig. 3C) (23). In contrast, the low symmetry of heme d₁ leads to numerous RR-active modes that are not related to the heme c bands. As a consequence, well established empirical relationships for iron porphyrins between band frequencies above 1300 cm⁻¹ and the oxidation, spin, and coordination state of iron (24) cannot be extended to heme d₁ and, therefore, most of its bands must remain unassigned. Remarkable exceptions are the bands at 1687 and 1725 cm⁻¹, which originate from the C=O stretching modes of the keto groups and the carboxyl function of the acrylic side chain, respectively. Note that differences between the spectrum in Fig. 3A and a spectrum of oxidized cd₁ published previously (25) are likely to be due to different excitation lines and temperatures.

FIGURE 3.

RR spectra of NirS_WT, NirS_ΔN, and semi-apo NirS measured with 442-nm excitation at 80 K. A, oxidized heme d₁ of NirS_WT. B, oxidized dihydro-heme d₁ of NirS_ΔN. C, oxidized heme c of semi-apo NirS. D, reduced heme c of semi-apo NirS. The spectra in A and B were obtained by subtracting the heme c contributions (spectra C and D) from the spectra of the (as-isolated) enzymes NirS_WT and NirS_ΔN, respectively. The spectra of the oxidized heme c (C) and reduced heme c (D) were generated by mutual spectra subtraction of the as-isolated (mainly oxidized) and dithionite-reduced semi-apo NirS because the spectrum of the pure oxidized semi-apo NirS cannot be measured directly because of photoreduction at low temperatures. Note that the ferrous heme c in semi-apo NirS undergoes an unprecedented reversible temperature-dependent transition from the low-spin to the high-spin configuration. The latter prevails at 80 K, as indicated by the characteristic marker bands at 1353, 1470, and 1607 cm⁻¹ (24, 32).

As already expected from the weaker absorbance at 442 nm for NirS_ΔN compared with NirS_WT (Fig. 2), the RR spectrum of the putative heme d₁ precursor suffers from a substantially weaker resonance enhancement compared with heme d₁ (Fig. 3B). Despite the relatively poor signal-to-noise ratio, the comparison of both spectra demonstrates that most of the bands of heme d₁ also have counterparts within ± 3 cm⁻¹ in the spectrum of the potential heme d₁ precursor, albeit with quite different relative intensities. Therefore, we conclude that the ground state structure of heme d₁ and its precursor are similar but not identical. This conclusion is consistent with the assignment of the heme d₁ precursor to a dihydro-heme d₁. Moreover, among the few bands of NirS_WT for which conjugate bands in NirS_ΔN are not immediately evident, the bands at 1611 and 1181 cm⁻¹ may be attributed to modes involving the C=C and C-(C=C) stretching coordinates, taking into account previous assignments of modes involving the vinyl substituents in protoporphyin IX (26–29). Furthermore, assuming a dihydro-heme d₁ in NirS_ΔN, the different intensity pattern compared with the spectrum of heme d₁ can readily be understood because the extension of the delocalized π-electron system affects both the absolute and relative RR intensities.

NirS_ΔN Contains Dihydro-Heme d₁

The results of the experiments described so far strongly suggest that the heme d₁ precursor dihydro-heme d₁ might indeed be bound in NirS_ΔN. To finally clarify this question, the non-covalently bound heme d₁ (or possibly dihydro-heme d₁) was extracted from the purified NirS_WT and NirS_ΔN as described under “Experimental Procedures.” Then, UV-visible absorption spectra of the oxidized and dithionite-reduced forms were recorded (Fig. 4, A and B). The absorption spectrum of the heme d₁ cofactor extracted from NirS_WT exhibits characteristic peaks at 408 and 683 nm and a shoulder at about 476 nm in the oxidized state, as reported previously (30, 31). In contrast, the spectrum of the oxidized form of the cofactor extracted from NirS_ΔN shows absorption maxima at 397 and 671 nm and also a shoulder at about 476 nm. After dithionite reduction, the absorption bands of the heme d₁ extracted from NirS_WT are shifted to 457 and 627 nm, whereas those of the cofactor extracted from NirS_ΔN are shifted to 447 and 611 nm. Again, these observations strongly suggest that the cofactor isolated from NirS_ΔN was indeed different from the heme d₁ extracted from NirS_WT. As described above, we suspected that the heme d₁ precursor dihydro-heme d₁ lacking the acrylic double bond might be present in NirS_ΔN. The UV-visible absorption spectra of the isolated cofactor are in line with this proposal because the characteristic heme d₁ absorption band at 683 nm in the oxidized state is shifted to a shorter wavelength by about 10 nm in the case of the cofactor from NirS_ΔN, and the corresponding band of heme d₁ in the reduced state at 627 nm is shifted by about 15 nm in this case. Such a hypsochromic shift would be expected for dihydro-heme d₁ because of the lacking double bond (6).

FIGURE 4.

Characterization of cofactors extracted from NirS_WT and NirS_ΔN. A and B, UV-visible absorption spectra of heme d₁ extracted from purified NirS_WT (d₁) and of dihydro-heme d₁ extracted from purified NirS_ΔN (dd₁) in the oxidized (as-isolated) (A) and dithionite-reduced (B) forms. Characteristic absorption bands are labeled. C and D, HR-ESI-MS analysis of the dihydro-heme d₁ extracted from NirS_ΔN (C) and of heme d₁ extracted from NirS_WT (D). The top panels show the experimental mass and isotopic pattern of the [M+H]⁺ ions, and the bottom panels show the calculated mass spectra of dihydro-heme d₁ (C) and heme d₁ (D), respectively. Note that the measured masses 711.57473, 712.57808, and 713.49640 in D originate from an uncharacterized impurity present in the heme d₁ sample. AU, absorption units.

Finally, we determined the exact mass of the cofactor extracted from NirS_ΔN by HR-ESI-MS. This method revealed a mass of 712.14675 for the [M+H]⁺ ion, which is indeed in accordance with the calculated mass (712.14621), isotopic pattern and chemical formula of dihydro-heme d₁ (Fig. 4C). For comparison, the mass of the heme d₁ extracted from NirS_WT was also determined by HR-ESI-MS. The measured value of 710.13075 for the [M+H]⁺ ion is in excellent agreement with the theoretical mass (710.13065), isotopic pattern and chemical formula of heme d₁ as expected (Fig. 4D). Moreover, the mass difference between the two cofactors isolated from either NirS_ΔN or NirS_WT corresponds to the two hydrogen atoms that are present in dihydro-heme d₁ (NirS_ΔN) but absent in heme d₁ (NirS_WT) because of the dehydrogenation of the propionate side chain of pyrrole ring D.

On the basis of all of the results described so far, we conclude that, in the absence of NirN in the bacterial cell, the formation of the acrylic double bond of heme d₁ does not take place and that dihydro-heme d₁ is inserted into NirS_ΔN. This novel finding led to the question whether NirN itself catalyzes the dehydrogenation reaction, which would be completely unexpected, or whether NirF catalyzes the double bond formation, as proposed previously, but with the help of NirN. These two possibilities were tested in vitro, and the results are described in the following section.

Purified, Recombinant NirN from P. aeruginosa Catalyzes the Conversion of Dihydro-heme d₁ to Heme d₁ in Vitro

To test whether NirN is able to catalyze the dehydrogenation of the propionate side chain of pyrrole ring D, yielding the acrylate side chain of heme d₁, the extracted dihydro-heme d₁ was mixed with purified NirN, and UV-visible absorption spectra were recorded at different time points after mixing. As shown in Fig. 5A, the absorption band of the free dihydro-heme d₁ at 671 nm disappeared upon addition of NirN, and a new absorption band at 609 nm was visible immediately after mixing. This new absorption feature most likely represents the binding of dihydro-heme d₁ to NirN. However, after 15 s of incubation, the spectrum had changed, and the absorption band at 609 nm had developed into a broad band at about 635 nm that did not change further during prolonged incubation. On the basis of this observation, we speculated that NirN might have catalyzed the formation of the acrylic double bond after the initial binding of the dihydro-heme d₁ to the enzyme. Therefore, we compared the final absorption spectrum of the NirN/dihydro-heme d₁ reaction mixture with the absorption spectra of a NirN/heme d₁ mixture in the oxidized and reduced states (Fig. 5B). Strikingly, the final spectrum of the NirN/dihydro-heme d₁ reaction mixture was identical to the spectrum of the NirN/heme d₁ mixture in the reduced state, both showing an absorption band at 635 nm. Furthermore, we then oxidized the NirN reaction mixture initially containing dihydro-heme d₁ by the addition of sodium persulfate, which resulted in a shift of the 635 nm absorption band to 626 nm. The obtained absorption spectrum was now identical to the spectrum of the NirN/heme d₁ mixture in the oxidized state (Fig. 5B). These results suggest that NirN is indeed able to catalyze the conversion of dihydro-heme d₁ to heme d₁.

FIGURE 5.

Binding of dihydro-heme d₁ to purified NirN and transfer to semi-apo NirS. A, UV-visible absorption spectra of the free dihydro-heme d₁ (dd₁), of purified NirN, and of the NirN/dihydro-heme d₁ mixture measured immediately after mixing the two components (NirN addition) and measured after 15 s of incubation. Characteristic absorption bands are labeled. B, UV-visible absorption spectra of NirN/heme d₁ (d₁) mixtures in the oxidized and reduced forms and of the NirN/dihydro-heme d₁ mixture after incubation for several minutes and subsequent oxidation with sodium persulfate. The spectra in A indicate that dihydro-heme d₁ first bound to NirN, resulting in a new absorption band at 609 nm, and was then transformed into a new species exhibiting an absorption band at 635 nm. Upon oxidation with sodium persulfate, this species showed an absorption band at 626 nm (B). The comparison of these spectra with those of the NirN/heme d₁ mixture (oxidized (ox.) and reduced (red.)) suggested that NirN catalyzed the transformation of dihydro-heme d₁ to heme d₁. C and D, UV-visible absorption spectra of the NirN/dihydro-heme d₁ mixture after several minutes of incubation and subsequent addition of NirS_s.a. (NirN + dd₁ + NirS_s.a.) and of NirS_s.a./heme d₁ and NirS_s.a./dihydro-heme d₁ mixtures in the oxidized (C) and reduced (D) forms. The comparison of the obtained spectra suggested that the cofactor that was transferred from NirN to NirS_s.a. after incubation of NirN with dihydro-heme d₁ was indeed the newly formed heme d₁. AU, absorption units.

To further substantiate this finding, we added NirS_s.a. to the NirN/dihydro-heme d₁ reaction mixture after the potential conversion of the precursor to heme d₁ was completed. Because it has been reported previously that heme d₁-loaded NirN is able to transfer the cofactor to NirS_s.a. (12), we assumed that this transfer should also take place in our reaction mixture. After several minutes of incubation with NirS_s.a., UV-visible absorption spectra were recorded. As shown in Fig. 5C, the absorption band at 626 nm potentially representing heme d₁-loaded NirN (oxidized) was shifted to 641 nm upon addition of NirS_s.a.. Indeed, the newly obtained spectrum was identical to the spectrum of heme d₁-containing NirS. Moreover, the same was true after the reduction of the cofactor with sodium dithionite (Fig. 5D). These observations clearly supported the idea that NirN catalyzed the conversion of dihydro-heme d₁ to heme d₁. The subsequent transfer of the formed heme d₁ to NirS_s.a. yielding holo-NirS took place as described previously (12).

Finally, to unambiguously demonstrate that heme d₁ was formed out of dihydro-heme d₁ by the action of NirN, we extracted the cofactor from the NirN/dihydro-heme d₁ reaction mixture after the reaction was completed and analyzed the formed reaction product by UV-visible absorption spectroscopy and HR-ESI-MS (Fig. 6). The extracted reaction product exhibited an absorption band at 683 nm identical to heme d₁. Moreover, a mass of 710.13043 was determined for the reaction product ([M+H]⁺), which is in agreement with the theoretical mass (710.13065) of heme d₁. Therefore, we concluded that NirN indeed catalyzed the formation of the acrylic double bond, therefore converting dihydro-heme d₁ to heme d₁.

FIGURE 6.

Characterization of the cofactor extracted from the NirN/dihydro-heme d₁ mixture. A, UV-visible absorption spectra of the cofactor extracted from the NirN/dihydro-heme d₁ reaction mixture after several minutes of incubation (NirN assay) and spectra of extracted heme d₁ (d₁) and dihydro-heme d₁ (dd₁) for comparison. Characteristic absorption bands are labeled. B, HR-ESI-MS analysis of the cofactor extracted from the NirN/dihydro-heme d₁ reaction mixture. The top panel shows the experimental mass and isotopic pattern of the [M+H]⁺ ion, and the bottom panel shows the calculated mass spectrum of heme d₁. Note that the measured masses 711.57397 and 712.57680 originate from an uncharacterized impurity present in the sample. AU, absorption units.

Purified, Recombinant NirF from D. shibae Binds Dihydro-Heme d₁ without Conversion to Heme d₁

The finding that NirN catalyzes the conversion of dihydro-heme d₁ to heme d₁ was completely unexpected because NirF was originally proposed to be responsible for this reaction. This proposal was made on the basis of the observation that the periplasmic NirF from P. pantotrophus was able to bind heme d₁ in vitro, and the obtained NirF-heme d₁ complex was interpreted to represent an enzyme-product complex (13). Therefore, we incubated purified, recombinant NirF from D. shibae with dihydro-heme d₁ and heme d₁, and the binding of the cofactors to the protein was followed by UV-visible absorption spectroscopy (Fig. 7). The spectrum of the NirF/heme d₁ mixture exhibits characteristic absorption bands at 429 and 629 nm in the oxidized state and at 448 and 627 nm in the reduced form, which is in agreement with the absorption features of heme d₁-containing NirF from P. pantotrophus reported previously (13). The spectrum of the NirF/dihydro-heme d₁ mixture displays absorption bands at 421 and 593 nm in the oxidized form and at 438 and 608 nm in the reduced form, showing that NirF is also able to bind the dihydro-heme d₁ precursor because the observed absorption features of the NirF/dihydro-heme d₁ mixture clearly differ from those of the free dihydro-heme d₁. More importantly, the absorption spectrum of the NirF/dihydro-heme d₁ mixture recorded directly after mixing the protein with the cofactor did not change further upon prolonged incubation, in contrast to what was observed for the NirN/dihydro-heme d₁ mixture (see above). Therefore, we concluded that NirF was able to bind dihydro-heme d₁ but did not convert it to heme d₁.

FIGURE 7.

Binding of heme d₁ and dihydro-heme d₁ to purified NirF. A and B, UV-visible absorption spectra of purified NirF, NirF incubated with heme d₁ (d₁), and NirF incubated with dihydro-heme d₁ (dd₁). Spectra were measured in the oxidized (A) and dithionite-reduced (B) forms. Characteristic absorption bands are labeled. AU, absorption units.

NirN Catalyzes the Double Bond Formation using Electron Bifurcation

As outlined above, our studies revealed a new and unexpected function of NirN during heme d₁ biosynthesis, namely that of the terminal dehydrogenase introducing the double bond into the propionate side chain of pyrrole ring D of dihydro-heme d₁ to yield the acrylate side chain of heme d₁. However, NirN must be a quite unusual dehydrogenase because it did not require the addition of any cofactor such as NAD⁺ or FAD/FMN typically employed by common dehydrogenases catalyzing two-electron oxidation reactions. Interestingly, in our initial NirN/dihydro-heme d₁ reaction described above, we observed that the produced heme d₁ was in the reduced Fe(II) state, although the substrate dihydro-heme d₁ entered the reaction mixture in the oxidized Fe(III) form. Therefore, it seemed that one electron of the two-electron oxidation remained on the reaction product itself. Nevertheless, the fate of the second electron was still unclear. In this context, it is important to note that NirN contains a covalently attached heme c cofactor (16), and, therefore, we speculated that the second electron might be transferred to the heme c. In the initial reaction mixture, no reduction of the heme c in NirN was observed. However, this reaction was performed under aerobic conditions, which might have led to a rapid reoxidation of any reduced heme c. Therefore, we repeated the NirN activity assay under anaerobic conditions in an anaerobic chamber. NirN in its oxidized form (i.e. containing Fe(III)-heme c) was mixed with oxidized Fe(III)-dihydro-heme d₁, and the reaction progress was followed by UV-visible absorption spectroscopy (Fig. 8). As before, a new absorption band at about 635 nm developed during the reaction, which represents the NirN-bound heme d₁ in its reduced Fe(II) form. Moreover, two new absorption peaks at 522 and 550 nm appeared simultaneously with the absorption band at 635 nm. These absorption features are indeed characteristic for the reduced Fe(II)-heme c in NirN (16). On the basis of this experiment, we conclude that NirN catalyzes the two-electron oxidation of dihydro-heme d₁ to heme d₁ using an electron bifurcation mechanism during which one electron ends up on the reaction product heme d₁ and the second electron is transferred to the heme c cofactor of the enzyme.

FIGURE 8.

NirN activity assay under anaerobic conditions. UV-visible absorption spectra of free dihydro-heme d₁ (dd₁), of purified NirN, and of the NirN/dihydro-heme d₁ reaction mixture incubated under anaerobic conditions in an anaerobic chamber. For the NirN/dihydro-heme d₁ reaction mixture, spectra were measured immediately after mixing the two components (light gray) and then every 15 s (from light to dark gray). Characteristic absorption bands are labeled. The increase or decrease of absorption bands during the reaction progress is indicated by arrows. AU, absorption units.

Novel Roles of NirN and NirF during Heme d₁ Biosynthesis and Insertion into NirS

On the basis of the experiments described in this study, we would like to assign a novel function to the NirN protein. So far, the role of NirN was unclear because nirN deletion strains did not show extreme growth phenotypes and still produced an active form of NirS, which led to the assumption that NirN was apparently not involved in heme d₁ biosynthesis. However, we observed that the deletion of the nirN gene in P. aeruginosa resulted in the production of a holo NirS with an obviously altered cofactor content (15). In this study, we could show that this form of holo NirS actually carried the heme d₁ precursor dihydro-heme d₁. Further, we showed that NirN is able to catalyze the conversion of dihydro-heme d₁ to heme d₁ in vitro. Therefore, we identified NirN as the terminal dehydrogenase catalyzing the last step of heme d₁ biosynthesis. Moreover, we propose that NirN represents a novel and unusual type of a periplasmic dehydrogenase that uses an electron bifurcation mechanism to catalyze a two-electron oxidation reaction carrying only a one-electron accepting redox cofactor (heme c). Such a mechanism is possible for NirN because its substrate/product itself is able to accept one electron.

Considering the finding that NirN catalyzes the last step of heme d₁ biosynthesis, the question arises what the function of NirF might be that was assumed previously to play the role now assigned to NirN. In this study, we observed that NirF is able to bind dihydro-heme d₁. Together with the previous observation that NirF from P. aeruginosa is a periplasmic, membrane-anchored lipoprotein that interacts with both NirN and NirS (14, 15), one might speculate that NirF could be involved in the uptake of dihydro-heme d₁ into the periplasm from a still unknown membrane transporter and then further transfer the precursor to NirN. NirN then catalyzes the formation of heme d₁, which is finally transferred to NirS, yielding the active holo form of the enzyme. Such a role of NirF in the uptake of the heme d₁ precursor into the periplasm is, of course, speculative at this stage and will require further research.