The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols

Significant recent advances have been made in studies of the major dissimilatory nitrate reductase (NarGHI) of Escherichia coli. This enzyme is a complex iron-sulfur ([Fe-S]) molybdoenzyme that oxidizes menaquinol or ubiquinol at a periplasmically oriented Q-site (Qp site), and reduces nitrate at a cytoplasmically-oriented molybdo-(bismolybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor. The Qp site, as well as two hemes, termed bL and bH, are localized in a hydrophobic diheme cytochrome b(Narl) that: (i) provides a conduit for electron-transfer from the periplasmically-oriented Qp-site; (ii) provides a membrane anchoring functionality for the membrane-extrinsic subunits (NarGH) that coordinate the Mo-bisMGD (NarG) and four [Fe-S] clusters (NarH); and (iii) helps ensure the separation of sites of H+-yielding and H+-consuming reactions such that enzyme turnover leads to the generation of a proton-electrochemical potential across the cytoplasmic membrane. This minireview focuses on recent advances and future prospects for the diheme cytochrome b subunit (Narl) of NarGHI.     

Primary structure characterization of a Rhodocyclus tenuis diheme cytochrome c reveals the existence of two different classes of low-potential diheme cytochromes c in purple phototropic bacteria

The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cytochrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal insertions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting different functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious homology to the low-potential diheme cytochromes characterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cytochrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes.     

QcrCAB operon of a nocardia-form actinomycete Rhodococcus rhodochrous encodes cytochrome reductase complex with diheme cytochrome cc subunit

Structural genes encoding quinol-cytochrome c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete Rhodococcus rhodochrous. QcrC and qcrA encode diheme cytochrome cc and the Rieske Fe-S protein, respectively, while the qcrB product is a diheme cytochrome b. These amino acid sequences are similar to those of Corynebacterium and Mycobacterium, the members of high G+C content firmicutes. The presence of diheme cytochrome cc subunit as a sole c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome c oxidase. The N-terminal half of the Rieske Fe-S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved. A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with Thermus, but not with low G+C firmicutes.     

The crystal structure of human S-adenosylmethionine decarboxylase at 2.25 A resolution reveals a novel fold.

BACKGROUND: S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine synthetic pathway, and a well-studied drug target. The AdoMetDC decarboxylation reaction depends upon a pyruvoyl cofactor generated via an intramolecular proenzyme self-cleavage reaction. Both the proenzyme-processing and substrate-decarboxylation reactions are allosterically enhanced by putrescine. Structural elucidation of this enzyme is necessary to fully interpret the existing mutational and inhibitor-binding data, and to suggest further experimental studies. RESULTS: The structure of human AdoMetDC has been determined to 2.25 A resolution using multiwavelength anomalous diffraction (MAD) phasing methods based on 22 selenium-atom positions. The quaternary structure of the mature AdoMetDC is an (alpha beta)2 dimer, where alpha and beta represent the products of the proenzyme self-cleavage reaction. The architecture of each (alpha beta) monomer is a novel four-layer alpha/beta-sandwich fold, comprised of two antiparallel eight-stranded beta sheets flanked by several alpha and 3(10) helices. CONCLUSIONS: The structure and topology of AdoMetDC display internal symmetry, suggesting that this protein may be the product of an ancient gene duplication. The positions of conserved, functionally important residues suggest the location of the active site and a possible binding site for the effector molecule putrescine.     

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.     

The crystal structure of cytochrome P460 of Nitrosomonas europaea reveals a novel cytochrome fold and heme-protein cross-link

We have determined the 1.8 A X-ray crystal structure of a monoheme c-type cytochrome, cytochrome P460, from Nitrosomonas europea. The chromophore possesses unusual spectral properties analogous to those of the catalytic heme P460 of hydroxylamine oxidoreductase (HAO), the only known heme in biology to withdraw electrons from an iron-coordinated substrate. The analysis reveals a homodimeric structure and elucidates a new c-type cytochrome fold that is predominantly beta-sheet. In addition to the two cysteine thioether links to the porphyrin typical of c-type hemes, there is a third proteinaceous link involving a conserved lysine. The covalent bond is between the lysine side-chain nitrogen and the 13'-meso carbon of the heme, which, following cross-link formation, is sp3-hybridized, demonstrating the loss of conjugation at this position within the porphyrin. The structure has implications for the analogous tyrosine-heme meso carbon cross-link observed in HAO.     

Covalent structure of the diheme cytochrome subunit and amino-terminal sequence of the flavoprotein subunit of flavocytochrome c from Chromatium vinosum.

The complete sequence of the 21-kDa cytochrome subunit of the flavocytochrome c (FC) from the purple phototrophic bacterium Chromatium vinosum has been determined to be as follows: EPTAEMLTNNCAGCHG THGNSVGPASPSIAQMDPMVFVEVMEGFKSGEIAS TIMGRIAKGYSTADFEKMAGYFKQQTYQPAKQSF DTALADTGAKLHDKYCEKCHVEGGKPLADEEDY HILAGQWTPYLQYAMSDFREERRPMEKKMASKL RELLKAEGDAGLDALFAFYASQQ. The sequence is the first example of a diheme cytochrome in a flavocytochrome complex. Although the locations of the heme binding sites and the heme ligands suggest that the cytochrome subunit is the result of gene doubling of a type I cytochrome c, as found with Azotobacter cytochrome c4, the extremely low similarity of only 7% between the two halves of the Chromatium FC heme subunit rather suggests that gene fusion is at the evolutionary origin of this cytochrome. The two halves also require a single residue internal deletion for alignment. The first half of the Chromatium FC heme subunit is 39% similar to the monoheme subunit of the FC from the green phototrophic bacterium Chlorobium thiosulfatophilum, but the second half is only 9% similar to the Chlorobium subunit. The N-terminal sequence of the Chromatium FC flavin subunit was determined up to residue 41 as AGRKVVVVGGGTGGATAAKYIKLADPSIEVTLIEP NTKYYT. It shows more similarity to the Chlorobium FC flavin subunit (60%) than do the two heme subunits. The N terminus of the flavin subunit is homologous to a number of flavoproteins, including succinate dehydrogenase, glutathione reductase, and monamine oxidase. There is no obvious homology to the Pseudomonas putida FC flavin subunit, which suggests that the two types of flavocytochrome c arose by convergent evolution. This is consistent with the dissimilar enzyme activities of FC as sulfide dehydrogenase in the phototrophic bacteria and as p-cresol methylhydroxylase in Pseudomonas. We also present a sequence "fingerprint" pattern for the recognition of FAD-binding proteins which is an extended version of the consensus sequence previously presented (Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107) for nucleotide binding sites.     

A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the QcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex

Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its E(m, 7) was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3'-downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe-S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b(6) show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b(6) and green sulfur-low G+C Gram-positive bacteria.     

Expression and characterization of the diheme cytochrome c subunit of the cytochrome bc complex in Heliobacterium modesticaldum

Heliobacterium modesticaldum is a Gram-positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b(6)f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc(1) complexes. One of the unique factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b(6)f complex or the monoheme cytochrome c(1) in the bc(1) complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c(4) proteins. The recombinant protein exhibits a single redox midpoint potential of +71mV versus NHE, which indicates that the two hemes have very similar protein environments.     

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.     

The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.     

Tuning a nitrate reductase for function. The first spectropotentiometric characterization of a bacterial assimilatory nitrate reductase reveals novel redox properties

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S](1+) cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly [4Fe-4S](1+) and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.     

Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens

Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.     

A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.     

X-ray structure of the membrane-bound cytochrome c quinol dehydrogenase NrfH reveals novel haem coordination

Oxidation of membrane-bound quinol molecules is a central step in the respiratory electron transport chains used by biological cells to generate ATP by oxidative phosphorylation. A novel family of cytochrome c quinol dehydrogenases that play an important role in bacterial respiratory chains was recognised in recent years. Here, we describe the first structure of a cytochrome from this family, NrfH from Desulfovibrio vulgaris, which forms a stable complex with its electron partner, the cytochrome c nitrite reductase NrfA. One NrfH molecule interacts with one NrfA dimer in an asymmetrical manner, forming a large membrane-bound complex with an overall alpha(4)beta(2) quaternary arrangement. The menaquinol-interacting NrfH haem is pentacoordinated, bound by a methionine from the CXXCHXM sequence, with an aspartate residue occupying the distal position. The NrfH haem that transfers electrons to NrfA has a lysine residue from the closest NrfA molecule as distal ligand. A likely menaquinol binding site, containing several conserved and essential residues, is identified.     

Refined structure of cytochrome c3 at 1.8 A resolution

The structure of cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki has been successfully refined at 1.8 A resolution. The crystallographic R factor is 0.176 for 9907 significant reflections. The isotropic temperature factors of individual atoms were refined and a total of 47 water molecules located on the difference map were incorporated in the refinement. The four heme groups are closely packed, with adjacent pairs of heme planes being nearly perpendicular to each other. The fifth and the sixth ligands of the heme iron atoms are histidine residues with N epsilon 2-Fe distances ranging from 1.88 A to 2.12 A. The histidine co-ordination to the heme iron is different for each heme group. The heme groups are all highly exposed to solvent, although the actual regions exposed differ among the hemes. The four heme groups are located in different environments, and the heme planes are deformed from planarity. The differences in the heme structures and their environments indicate that the four heme groups are non-equivalent. The chemical as well as the physical properties of cytochrome c3 should be interpreted in terms of the structural non-equivalence of the heme groups. The characteristic secondary structural non-equivalence of the heme groups. The characteristic secondary structures of the polypeptide chain of this molecule are three short alpha-helices, two short beta-strands and ten reverse turns.     

The haem-copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II

The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem-copper types. The haem-copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa3 haem-copper oxygen reductase. The expression of D. vulgaris haem-copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem-copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem-copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.