Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex.

Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex. Recent structure-based spectropotentiometric studies are shedding light on the catalytic mechanism of NrfA; however, electron input into the enzyme has not been addressed biochemically. This study reports the first purification of NrfB, a novel 20-kDa pentaheme c-type cytochrome encoded by the nrfB gene that follows the nrfA gene in many bacterial nrf operons. Analyses by gel filtration demonstrated that NrfB purifies as a decaheme homodimer. Analysis of NrfB by UV-visible and magnetic circular dichroism spectroscopy demonstrates that all five NrfB ferric heme irons are low spin and are most likely coordinated by two axial histidine ligands. Spectropotentiometry revealed that the midpoint redox potentials of five ferric hemes were in the low potential range of 0 to -400 mV. Analysis by low temperature EPR spectroscopy revealed signals that arise from two classes of bis-His ligated low spin hemes, namely a rhombic trio at g(1,2,3) = 2.99, 2.27, and 1.5 that arises from two hemes in which the planes of histidine imidazole rings are near-parallel and a large g(max) signal at g = 3.57 that arises from three hemes in which the planes of the histidine imidazole rings are near-perpendicular. NrfB was also overexpressed as a recombinant protein, which had similar spectropotentiometric properties as the native protein. Reconstitution experiments demonstrated that the reduced decaheme NrfB dimer could serve as a direct electron donor to the oxidized decaheme NrfA dimer, thus forming a transient 20-heme [NrfB](2)[NrfA](2) electron transfer complex.

The reduction of nitrate to ammonium in the periplasm of Escherichia coli involves two enzymes, periplasmic nitrate reductase (NapA) and periplasmic cytochrome c nitrite reductase (NrfA). The process is found in many enteric bacteria and may be important for anaerobic nitrate and nitrite respiration at low nitrate concentrations (1,2). The NrfA protein catalyzes the six-electron reduction of nitrite to ammonium but can also catalyze the five-electron reduction of NO and two-electron reduction of hydroxylamine, both of which may be bound intermediates in the catalytic cycle for nitrite reduction (3,4). Indeed a possible physiological role of the enzyme in NO detoxification has recently been suggested (5).
The E. coli NrfA protein is a 52-kDa pentaheme cytochrome in which four hemes are covalently bound to the conventional motif CXXCH. The fifth heme is attached to the novel CXXCK motif that is essential for catalysis (6). The crystal structures of cytochrome c nitrite reductase from the sulfur-reducing bacterium Sulfurospirillum deleyianum, the closely related rumen bacterium Wolinella succinogenes, and the enteric bacterium E. coli have been determined recently (4,(7)(8)(9). In all three structures NrfA crystallized as a homodimer, with the hemes within each monomer closely packed to form arrangements of near-parallel and near-perpendicular heme pairs. In the absence of substrate, the NrfA active site heme displays a distal lysine ligand and proximal water or hydroxide ligand (8,9).
Analysis of the organization of nrfA gene clusters from a range of bacteria reveals that they can be divided into two groups. In one group, which includes W. succinogenes and S. deleyianum, nrfA clusters with an adjacent gene, nrfH, that encodes a membrane-anchored tetraheme quinol dehydrogenase of the NapC family (10 -13). In the second group, which includes E. coli, nrfA clusters with genes encoding a putative periplasmic pentaheme cytochrome (nrfB), a periplasmic (4 ϫ [4Fe4S]) ferredoxin (nrfC) and an integral membrane putative quinol dehydrogenase (nrfD) (3,5). Clearly, electron transfer from quinol to the NrfA in the different groups is distinct. The different protein-protein and cofactor-cofactor interactions implicit in this situation may be reflected by insertions and deletions in loop regions of the polypeptide chain in the two subgroups that can be identified in primary and tertiary structure analyses (9). Despite the importance of periplasmic nitrite reduction to ammonium in enteric bacteria and the developing structure-informed biochemical understanding of the enzyme that catalyzes this process, the nature of electron delivery has never been addressed biochemically. In this paper we present the first purification and spectropotentiometric characterization of NrfB and demonstrate its competence as an electron donor to NrfA, with which it must transiently form a 20-heme [NrfB] 2 [NrfA] 2 electron transfer complex. levels during anaerobic growth in the presence of nitrate (14). Cultures of strain LCB2048 were grown without aeration overnight at 37°C in minimal salts medium (14) supplemented with 0.4% glycerol, 20 mM nitrate, kanamycin (25 g/ml), and spectinomycin (25 g/ml). Initial cultures (250 ml) were inoculated with 0.5 ml of an overnight culture grown aerobically on Luria-Bertani broth medium. The 250-ml culture was successively transferred to 2, 6, and 100 liters of fresh medium.
Purification of Native E. coli NrfB and NrfA-Periplasmic proteins were extracted from the harvested cells as described previously (14). The enzyme was precipitated from the periplasm using 65% saturated ammonium sulfate and resuspended in 50 mM Tris-HCl, pH 7. The precipitate was dialyzed and applied to an anion exchange Q-Sepharose column (35 ϫ 3 cm) equilibrated with 50 mM Tris-HCl, pH 7. The column was developed using a linear gradient of 0 -200 mM NaCl. NrfB eluted at ϳ100 mM and NrfA eluted at ϳ80 mM NaCl. The NrfB and NrfA fractions were applied separately to a Superdex G-75 HiLoad 16/60 fast protein liquid chromatography column equilibrated with 50 mM Tris-HCl, pH 7. NrfB and NrfA were then further purified on an anion exchange Dionex column (0.9 ϫ 25 cm) equilibrated with 50 mM Hepes, pH 7, using a SPRINT system. The column was developed using a 0-100 mM NaCl linear gradient, and NrfA eluted at 60 mM NaCl. Protein concentration was determined by the BCA method using bovine serum albumin as a protein standard. The NrfA and NrfB were judged pure on the basis of Coomassie Blue and heme-stained SDS-PAGE ( Fig. 1).
Expression of Recombinant nrfB-The nrfB gene encoding NrfB was amplified from E. coli strain JM109 genomic DNA by PCR using genespecific primers (5Ј-ATGAGAGTATTACGTTCGTT-3Ј and 5Ј-TCATG-GCTGCTCCTTAAGCA-3Ј) and the proofreading polymerase pwo. The resulting DNA fragment was purified using a commercial gel extraction kit (Qiagen) and inserted by blunt end ligation into the EcoRV site of the plasmid pETBlue-1 (Novagen) to place the recombinant nrfB under the control of the IPTG 1 -inducible T7 promoter. Recombinant plasmids were identified by blue-white screening, and the orientation of nrfB inserts was determined by restriction digests. Suitable recombinant plasmids were transformed into the expression host E. coli BL21(DE3)(pEC86). The accessory plasmid pEC86 provided constitutive expression of the cytochrome c maturation (ccm) gene cluster, thus allowing expression of recombinant c-type cytochromes under aerobic conditions. E. coli BL21(DE3)(pEC86)(pNrfB) was grown on Luria-Bertani medium plus ampicillin (100 g ml Ϫ1 ) and chloramphenicol (30 g ml Ϫ1 ) at 37°C with aeration to the mid-exponential phase prior to induction with 1 mM IPTG. Cells were harvested by centrifugation 16 h after induction.
Purification of Recombinant E. coli NrfB-Whole cell extracts of induced BL21(DE3)(pEC86)(pNrfB) were generated by sonication of the harvested and washed cells in 10 mM Tris-HCl, pH 8. NrfB, as indicated by a red coloration, was precipitated by 10 -20% ammonium sulfate and then resuspended in and dialyzed against 10 mM Tris-HCl, pH 8. This dialyzed sample was then further separated on a DEAE-Sepharose column equilibrated in the same buffer by using a linear gradient of 0 -1 M NaCl over 5 column volumes. NrfB was found to elute at 300 mM NaCl.
Activity Assays-Nitrite reductase activity was measured spectrophotometrically by substrate-dependent oxidation of reduced methyl viologen (⑀ 600 nm ϭ 13,700 M Ϫ1 cm Ϫ1 ). Assays (3.5-ml final volume) were performed by mixing at 25°C in anaerobic cuvettes containing 1 mM methyl viologen, 2 mM CaCl 2 , 50 mM Hepes, pH 7, and either nitrite or hydroxylamine. Methyl viologen was reduced by the addition of sodium dithionite, and turnover was initiated by the addition of NrfA (0.14 g/ml).
MCD and EPR Spectroscopy-Perpendicular and parallel mode EPR measurements were performed with a Bruker EMX spectrometer equipped with an Oxford ESR-9 liquid helium cryostat and a dual mode cavity with microwave frequencies of 9.65 and 9.35 GHz for the perpendicular and parallel modes, respectively. MCD experiments were recorded on either a circular dichrograph, JASCO J-810, for the wavelength range 280 -1000 nm and a JASCO J-730 for the range 800 -2000 nm. Samples were exchanged into deuterated buffer and placed in quartz cuvettes within an Oxford Instruments SM1 6T superconducting solenoid with an ambient temperature bore for room temperature measurements.
Visible Absorption Spectra and Mediated Redox Potentiometry-Ab-sorption spectra were collected using an Amicon SLM DW2000 spectrophotometer. The UV-visible spectra and redox titration of soluble NrfB was carried out at 25°C in 100 mM Tris-HCl, pH 8, and 100 mM NaCl. Mediated redox potentiometry was performed as described previously (9). Dithionite and ferricyanide were used as reductant and oxidant, respectively. Redox mediators were phenazine methosulfate, phenazine ethosulfate, diaminodurene, 4-hydroxynapthoquinone, 5-anthraquinone 2-sulfonate, 6-anthraquinone 2,6-disulfonate, and benzylviologen (at a final concentration of 20 M). A saturated quinhydrone solution (E pH7 ϭ ϩ295 mV) was used to calibrate the electrode. All potentials quoted are with respect to the normal hydrogen electrode. Redox titrations were fitted with a variable number of n ϭ 1 Nernstian components using a customized program that allowed the midpoint redox potential, the number of redox components, and the contribution of each component to float as appropriate.
Analytical Methods-Native PAGE was performed using commercially prepared 12% gels (Gradipore Ltd.) to separate purified samples of NrfA and NrfB under non-denaturing conditions. After running, the gels were soaked in standard SDS-containing running buffer, and the proteins were visualized by staining for covalently bound heme using the peroxidase assay. Analytical gel filtration was carried out on both S-75 and S-200 gel filtration columns (Amersham-Pharmacia) in 10 mM Tris-HCl, pH 8, and 200 mM NaCl. Samples were loaded in the same buffer from a 100-l injection loop, and the columns were run at a flow rate of 1 ml min Ϫ1 . Elution profiles were recorded by monitoring the A 280 nm of the eluate.
Reconstitution of NrfB with NrfA-Purified NrfB (0.4 M) in 10 mM Tris-HCl was reduced by the addition of an equimolar amount of sodium dithionite in a sealed, anaerobic, 1-ml cuvette. A catalytic amount of NrfA (5 nM) and 1 mM nitrite were added by injection from anaerobic stock solutions. The transition of NrfB from a reduced to oxidized state was monitored by recording changes in the absorbance at 552 nm over time.

RESULTS
Purification and Quaternary Structure of NrfB-Purified NrfB migrated as a single, ϳ20-kDa band on Coomassie-stained and heme-stained SDS-PAGE gels (Fig 1, A and B). By comparison with 52-kDa NrfA, the 20-kDa NrfB stained poorly with Coomassie Blue. However, NrfB and NrfA stained comparably for heme ( Fig 1B), which reflects the presence of five hemes per polypeptide in both proteins. The purification procedure for NrfB yielded ϳ0.5 mg of protein at this level of purity from a 100-liter culture. Analysis of the NrfB pro-protein using the SignalP program suggests that the signal peptide is cleaved at position Ala 34 -Ser 35 . This would give a molecular mass of 17,549, which increases to 20,579 when five covalently attached apo-heme groups are added, a figure consistent with the SDS-PAGE analysis. Analysis by gel exclusion chromatography yielded a molecular weight of 33 kDa (Fig. 1C). This indicates that native NrfB is a decaheme homodimer in solution.
UV-Visible Properties of NrfB-The oxidized and reduced UV-visible spectrum of NrfB ( Fig. 2A) are typical of c-type cytochromes. The oxidized spectrum has maxima at 409 and 532 nm, which are characteristic of low spin ferric hemes. No features between 600 -650 nm, characteristic of a ligand-metal charge transfer (LMCT) band of a high spin heme, were observed in freshly prepared samples. It was noted that such a signal did develop on prolonged storage (over a period of weeks) at 4°C, which presumably reflected the lability of an axial heme ligand on one or more of the hemes, and such samples were not used for further spectroscopic or potentiometric analyses. The dithionite-reduced NrfB spectrum exhibits the Soret, ␤, and ␣ bands at 420.5, 523.5 and 552 nm, which are characteristic of low spin ferrous c-type hemes (Fig 2A).
Reconstitution of NrfB with NrfA-In equimolar mixtures, NrfA and NrfB did not co-elute when passed down gel exclusion columns under a range of conditions and also did not co-migrate upon subjection to native PAGE, showing that they do not form a tight complex. However, upon the addition of nitrite (1 mM) to dithionite-reduced NrfB (0.5 M) in the presence of catalytic amounts of NrfA (5 nM), a rapid oxidation of NrfB was observed as evidenced by a decrease in absorbance of the reduced NrfB ␣ peak at 552 nm (Fig. 2B). The initial rate of oxidation of NrfB under the experimental reaction conditions was 100 electrons s Ϫ1 . Spectra collected at the end of the experiment confirmed that full re-oxidation of NrfB had occurred. This finding indicates that, in the presence of the nitrite reductase NrfA, electrons can flow from NrfB via NrfA to nitrite. The full oxidation of NrfB via the 100-fold sub-stoichiometric NrfA shows that the oxidized NrfA dimer must interact transiently with a number of molecules of the reduced NrfB dimer during the time course of the experiment. In control experiments, oxidation of ferrous NrfB was not observed when nitrite was added to NrfB in the absence of NrfA, but NrfB oxidation was observed when NrfA was subsequently added (not shown). Not surprisingly for oxidation of a decaheme complex, the oxidation kinetics of NrfB were polyphasic, and, although not analyzed in detail, ionic strength appeared to have only marginal effect within the range 0 -0.5 M NaCl. This observation suggested that the dominant forces stabilizing the interaction are not electrostatic in nature.
Spectropotentiometric Properties of NrfB-Spectra of NrfB were collected at a number of potentials in the range of 400 mV to Ϫ400 mV. Plots of the increase in the absorbance change (measured at 552Ϫ700 nm) as a function of potential can be most simply fitted to five independently titrating (n ϭ 1) low spin Nernstian components, each of which contribute approximately equally to the total reduced absorption peak (Fig 3A). The best fit to the data gave midpoint potentials at Ϫ63, Ϫ221, and Ϫ259 mV for three hemes and at Ϫ129 mV for two hemes with iso-midpoint potentials. Inspection of the spectra collected at each potential did not reveal any changes in the position of the max (Fig 3B; note that at low potential, spectral features in the region of 600 -650 nm become obscured by the absorption of reduced methyl viologen, which is present as a redox mediator) Magnetic Circular Dichroism of NrfB-The UV-visible MCD spectrum of NrfB exhibited an intense, derivative-shaped band centered at ϳ400 nm in the Soret region (Fig 4A). When normalized for the presence of five hemes, this derivative had a peak to trough intensity of ϳ150 M Ϫ1 cm Ϫ1 tesla Ϫ1 heme Ϫ1 . This value is consistent with that observed for other low spin heme proteins (15,16). Thus, the intensity of the MCD Soret band derivative is consistent with the presence of five low spin hemes in NrfB. This result is also supported by the ␣,␤-MCD  7. B, NrfA-and nitrite-dependent re-oxidation NrfB. NrfB (0.5 M) was pre-reduced using sodium dithionite. Absorbance was measured at 552 nm, and NrfA (5 nM) and nitrite (1 mM) was added as indicated. Spectra were recorded at the start and end of the experiments to confirm that the NrfA was fully reduced at the start and fully oxidized at the end. Black line, no added NaCl; gray line, plus 500 mM NaCl. bands at 500 -600 nm, which are typical of low spin heme. No signals characteristic of high spin species in the region of 600 -650 nM were apparent. The charge transfer band for low spin ferric heme occurs in the near-infrared (NIR) region of the electronic absorption spectrum in the region of 800 -2500 nm. This LMCT band is rarely detected by absorption spectroscopy but can be readily detected by MCD spectroscopy, with the peak wavelength being an excellent indicator of the axial ligands to the heme iron (15,22). In NrfB, a broad positive LMCT band was observed at 1500 nm with a vibrational side band at lower wavelength (1200 -1300 nm). This observation is characteristic of low spin bis-His coordinated ferric heme ( Fig  4B). The 1500-nm band displays a broadening on its low energy side (ϳ1520 nm), which reflects the fact that five hemes in different protein environments contribute to this signal. At the max of the LMCT NIR MCD bands, low spin bis-His hemes characteristically have a ⌬⑀ of between 0.8 and 1.2 M Ϫ1 cm Ϫ1 tesla Ϫ1 . The intensity of the NrfB LMCT band when normalized for five hemes is 1.05 M Ϫ1 cm Ϫ1 tesla Ϫ1 and is thus again consistent with the presence of five low spin hemes.
EPR Spectroscopy of NrfB-The low temperature frozen solution perpendicular mode X-band EPR spectrum of air-oxidized NrfA collected at a temperature of 10 K and with microwave power of 2 milliwatts is complex (Fig 5A). The EPR signals at g Ϸ 2.99, 2.27, and 1.5 are typical for the g z , g y , and g x components of rhombic Fe(III) signals arising from low spin bis-His-ligated Fe(III) hemes in which the planes of the imidazole rings are near-parallel (23). Quantification of this signal by integration against a 1 mM copper EDTA standard gave a nearest integer value of 2 spin NrfB Ϫ1 . The second heme signal is a weak "large g max " signal at g z Ϸ 3.57 (23). Such signals have low intensities with broad line widths and are therefore difficult to detect and quantify (24). However, the feature becomes better resolved at higher microwave powers (Fig. 5B). Spin quantification yielded a value of 3 spin NrfB Ϫ1 . The large g max signal is likely to arise from NrfB hemes in which the planes of the two imidazole rings of the His ligands are near-perpendicular. There was no strong signal at g ϭ 6, characteristic of magnetically isolated high spin heme, which was consistent with the absence of high spin heme features in the UV-visible and MCD spectra. No broad features at g ϳ 12 or g ϳ 3.5, characteristic of spincoupled hemes (S ϭ 1/2 and S ϭ 5/2) that have been observed in NrfA (9), were observed in either the parallel or the perpendicular mode spectra of NrfB. The contribution of two hemes to the rhombic signal and three hemes to the large g max signal demonstrates that all five NrfB hemes are detectable and that none are rendered EPR silent through magnetic interactions with each other.
Expression of Recombinant nrfB-In NrfA, heme 1 is attached to a CXXCK motif, and the NrfEFG assembly proteins encoded within the nrf operon are required for heme attachment to this unusual motif in which the Lys provides the proximal heme iron ligand (10). NrfB heme 1 is attached to a CXXCHK motif, but it is most likely, although not certain, that the His rather than the Lys is the proximal heme iron ligand. To assess the requirements for the maturation of NrfB, the nrfB gene was cloned into the expression vector pETBlue-1 to yield pNRFB6 and co-expressed with the cytochrome maturation (ccm) genes (encoded on pEG86) that encode the proteins required for the attachment of hemes to CXXCH motifs. Expression under aerobic conditions, where the expression of the native nrfEFG genes is suppressed, resulted in synthesis of holo-NrfB in the soluble fraction, which was maximal following induction with IPTG in the presence of the ccm genes (pEG86) (Fig 1D, lanes 6 -10). Fractionation of cells established that the recombinant NrfB was localized in the periplasmic fraction, and purification yielded 100 mg of pure protein from 100 liters of culture, a 200-fold higher yield than that for the native protein. Analysis of this recombinant protein, using the techniques applied for native NrfA described above, showed it to be competent as an electron donor to NrfA and to have similar spectroscopic and redox properties as the native protein. An illustration of this finding is shown in the EPR spectrum (Fig 5, C and D). As for native NrfB, both high g max (g Ϸ 3.57) and rhombic signals (g Ϸ 2.99, 2.27, and 1.5) are present, indicative of hemes with nearparallel and near-perpendicular imidazole ligands, and they integrate to yield a 3:2 ratio. Efforts to express cloned nrfA under similar conditions were unsuccessful. This result indicates that the products of nrfEFG, which are required for correct folding and heme insertion into NrfA, are not required for NrfB. This observation is consistent with the spectroscopic evidence that, unlike NrfA, all five NrfB hemes are low spin bis-His coordinated species. DISCUSSION This work has provided the first purification and spectropotentiometric characterization of the NrfB cytochrome from any source. The genes for these novel pentaheme c-type cytochromes are only found in gene clusters that also encode the NrfA pentaheme nitrite reductase in enteric members of the ␥-proteobacteria. The nrfA and nrfB genes are located contiguously, implicating a role for NrfB as an electron donor to NrfA, and the first biochemical evidence for this implication is now provided. The pentaheme NrfA nitrite reductase polypeptide is ϳ50 kDa and, thus, has a heme/protein ratio of ϳ1 heme per 10 kDa. By comparison, the NrfB cytochrome is very small, having a polypeptide mass of ϳ17 kDa and, hence, a heme/protein ratio of only ϳ1 heme per 3.4 kDa. This will lead to a very close packing of the five hemes that will most likely allow for rapid electron transfer through the protein to the redox partner in a manner also suggested for small tetraheme cytochromes (STCs) such as the Shewanella oneidensis STC (18). The MCD analysis of NrfB is consistent with all five hemes being low spin hexacoordinate ferric species. The NIR MCD peak at 1500 nm suggests that these hemes all have bis-histidinyl ferric iron axial ligation, which would require 10 histidine residues to be present in the NrfB polypeptide chain. Sequence alignments of NrfB members reveal 11 conserved His residues, five in the CXXCH heme binding motifs that provide the proximal ferric iron ligands and six elsewhere that are candidates for the five distal ferric iron ligands (Fig. 6). A conserved methionine residue identified in the alignment can be excluded as a distal heme ferric iron ligand by the MCD, because a His-Met ligand pair would give rise to a NIR MCD peak of ϳ1800 nm, and no such peak was apparent. However, it should be noted that a His-Lys ligand pair can also give rise to a peak in the 1500 nm region of the NIR MCD spectrum (17), and there are conserved Lys residues in NrfB (Fig. 6), one of which is close to the first heme binding motif. Given that the active site heme iron in the NrfA nitrite reductase has a Lys proximal ligand, the possibility of a Lys ligand to a ferric heme iron in NrfB cannot be excluded. However, if a low spin ferric heme iron possesses a His-Lys axial ligand pair, it characteristically has a sharp signal at g ϳ 3.5 in the EPR spectrum (17). Such a signal was not apparent in the NrfB EPR spectrum. If, as in NrfA, there is a Lys-coordinated heme that does not have a second proteinderived ligand, it is usually a high spin species. Again, no evidence for such a species was apparent in freshly prepared samples. Finally, the requirement for only the standard cytochrome c maturation genes to facilitate the synthesis of recombinant holo-NrfB during anaerobic growth also argues against an unusual Lys coordination to the heme iron. Thus, taking the MCD, EPR, sequence alignment, and expression experiments together, we favor a model for NrfB in which all five ferric hemes are bis-histidinyl-coordinated low spin species attached to CXXCH heme binding motifs. This model distinguishes the pentaheme core of NrfB from that of its enzymatic redox partner, NrfA, in which only four or the five hemes are low spin, with the fifth heme iron being the high spin Lys-coordinated species to which nitrite binds and at which catalysis takes place. The absence of such a site in NrfB was consistent with the failure to observe any direct nitrite reduction by the reduced protein in the absence of NrfA.
The electron-donating properties of the imidazole ring nitrogen ligands stabilize the ferric state of heme iron with bis-His axial coordination (17). Consequently, bis-His-ligated hemes usually have low Fe(III)/Fe(II) midpoint redox potentials. The reduction of all five NrfB hemes in the region of 0 to Ϫ400 mV is thus consistent with bis-His heme iron coordination. The low equilibrium midpoint potentials of these hemes overlap with those of the hemes of NrfA (one at Ϫ37 mV, two at Ϫ107 mV, and two at Ϫ323 mV), such that a simple exergonic movement of electrons in an energetically favorable manner from one electron carrier to the next cannot be envisaged. Rather, the electron transfer to the active site of NrfA through the NrfAB hemes must involve both exergonic and endergonic electron FIG. 5. Perpendicular mode X-band EPR spectra of airoxidized native and recombinant NrfB. Spectra were collected at a temperature of 10 K and microwave power of 2 milliwatts (A and C) and a temperature of 10 K and microwave power of 40 milliwatts (B and D). For both spectra, the modulation amplitude was 1 millitesla (mT), the microwave frequency was 9.67 GHz, and the NrfB concentration was 40 M in 0.05 M Hepes, pH 7. A and B, native NrfB. C and D, recombinant NrfB. The features at g ϭ 3.57, 2.99, 2.27, and 1.5 are discussed under "Results." The feature at g ϭ 4.3 arises from adventitious iron. The gap in the data at ϳ325-350 milliteslas is due to removal of a cavity signal. transfer, with rapid flux ensured by close interaction of the hemes within the protein milieu. NrfB has been shown, through the use of analytical gel filtration, to occur as a decaheme homodimer in solution. As NrfA is also a decaheme homodimer, this finding is consistent with the formation of a transient [NrfA] 2 [NrfB] 2 tetrameric 20-heme electron transfer complex. Examination of the structure of NrfA (9) suggests that the NrfB dimer would dock in a large pocket at the base of the protein with heme 2 and/or heme 5 being potential electron input sites.
In the NrfA structure, the hemes are organized as parallel or perpendicular heme pairs in which the tetrapyrrole rings can be as close as 4 Å. The high heme:protein ratio of NrfB strongly suggests that such closely interacting hemes will be a feature of this cytochrome group. The NrfB primary structure does not have significant homology with any structurally defined multiheme cytochrome in the current data bases. However some features are suggestive of parallel heme pair organization. In the structurally defined STC (18) and the tetra-heme domains of flavocytochrome c fumarate reductases (19,20) a broadly conserved primary structure feature can be identified that binds a parallel heme pair, CXXCHX 9 -17 HXXHX 3-6 CXXCH-XXH (Fig. 5), where the second His between the two CXXCH motifs provides a distal ligand to the heme covalently attached to the second CXXCH motif. The His 9 -17 amino acids after the first CXXCH motif and the His two amino acids after the second CXXCH motif provide distal ligands for hemes that are not part of the heme pair. This feature, with similar amino acid spacing, can be identified in NrfB with the sequence CXXCH-X 11 HXXHX 5 CXXCHXXXH (Fig. 6). In STC and flavocytochrome c, two of the four hemes have near-perpendicular His ligands, the other two have near-parallel His ligands, and all of the hemes titrate in the low potential domains, features that are in common with that observed for NrfB in this study. It is notable that one STC also has a CXXCHK motif at the first heme binding site similar to that of NrfB, and from best fit alignments it appears that NrfB and STC may have diverged in evolution through a deletion/insertion of the second heme binding region of the polypeptide (Fig. 6) Another group of multiheme cytochromes that merit consideration in the context of NrfB are the periplasmic decaheme cytochromes (e.g. MtrA) of the Fe(III)-respiring bacteria of the Shewanella genus. These decaheme cytochromes have only recently been characterized spectropotentiometrically (21). They are ϳ40 kDa proteins in which the 10 hemes are bound to the polypeptide in two pentaheme segments. Given that the decaheme NrfB dimer is also ϳ40 kDa, the possibility that the decaheme cytochromes arose from gene duplication and fusion of an nrfB-like gene is apparent. Indeed, the arrangement of the CXXCH motifs in the first pentaheme segment of MtrA is rather similar to that of NrfB, including a CXXCHX 9 -12 H-X 0 -3 HX 3-6 CXXCHXXH motif between heme binding sites 3 and 4 and the CXXCHK motif at heme binding site 1. Spectropotentiometric analysis of S. oneidensis decaheme MtrA reveals that, like the decaheme NrfB dimer, all ten hemes exhibit bis-His axial coordination with both near-parallel and nearperpendicular imidazole ring geometries and titrate in the range 0 to Ϫ400 mV (21).
In conclusion then, this paper has presented the first purification and characterization of NrfB from any organism and has provided biochemical evidence for the role of this protein as a direct electron donor to the NrfA nitrite reductase with which it must form a transient 20-heme [NrfB] 2 [NrfA] 2 complex. The spectropotentiometric and primary structure analyses of NrfB suggest that this pentaheme cytochrome may be the basic molecular building block from which larger decaheme cytochromes have evolved with other functions in bacterial respiratory systems. The high yielding purification procedure established for the recombinant protein will then provide large quantities of material required for future structure-function studies on this intriguing protein.