Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.     

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.     

Covalent structure of the flavoprotein subunit of the flavocytochrome c: sulfide dehydrogenase from the purple phototrophic bacterium Chromatium vinosum.

The amino acid sequence of the flavoprotein subunit of Chromatium vinosum flavocytochrome c-sulfide dehydrogenase (FCSD) was determined by automated Edman degradation and mass spectrometry in conjunction with the three-dimensional structure determination (Chen Z et al., 1994, Science 266:430-432). The sequence of the diheme cytochrome c subunit was determined previously. The flavoprotein contains 401 residues and has a calculated protein mass, including FAD, of 43,568 Da, compared with a mass of 43,652 +/- 44 Da measured by LDMS. There are six cysteine residues, among which Cys 42 provides the site of covalent attachment of the FAD. Cys 161 and Cys 337 form a disulfide bond adjacent to the FAD. The flavoprotein subunit of FCSD is most closely related to glutathione reductase (GR) in three-dimensional structure and, like that protein, contains three domains. However, approximately 20 insertions and deletions are necessary for alignment and the overall identity in sequence is not significantly greater than for random comparisons. The first domain binds FAD in both proteins. Domain 2 of GR is the site of NADP binding, but has an unknown role in FCSD. We postulate that it is the binding site for a cofactor involved in oxidation of reduced sulfur compounds. Domains 1 and 2 of FCSD, as of GR, are homologous to one another and represent an ancient gene doubling. The third domain provides the dimerization interface for GR, but is the site of binding of the cytochrome subunit in FCSD. The four functional entities, predicted to be near the FAD from earlier studies of the kinetics of sulfite adduct formation and decay, have now been identified from the three-dimensional structure and the sequence as Cys 161/Cys 337 disulfide, Trp 391, Glu 167, and the positive end of a helix dipole.     

Engineering of proteolytically stable NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase (CPR) is a membrane-bound flavoprotein that interacts with the membrane via its N-terminal hydrophobic sequence (residues 1-56). CPR is the main electron transfer component of hydroxylation reactions catalyzed by microsomal cytochrome P450s. The membrane-bound hydrophobic domain of NADPH-cytochrome P450 reductase is easily removed during limited proteolysis and is the subject of spontaneous digestion of membrane-binding fragment at the site Lys56-Ile57 by intracellular trypsin-like proteases that makes the flavoprotein very unstable during purification or expression in E. coli. The removal of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase results in loss of the ability of the flavoprotein to interact and transfer electrons to cytochrome P450. In the present work, by replacement of the lysine residue (Lys56) with Gln using site directed mutagenesis, we prepared the full-length flavoprotein mutant Lys56Gln stable to spontaneous proteolysis but possessing spectral and catalytic properties of the wild type flavoprotein. Limited proteolysis with trypsin and protease from Staphylococcus aureus of highly purified and membrane-bound Lys56Gln mutant of the flavoprotein as well as wild type NADPH-cytochrome P450 reductase allowed localization of some amino acids of the linker fragment of NADPH-cytochrome P450 reductase relative to the membrane. During prolong incubation or with increased trypsin ratio, the mutant form showed an alternative limited proteolysis pattern, indicating the partial accessibility of another site. Nevertheless, the membrane-bound mutant form is stable to trypsinolysis. Truncated forms of the flavoprotein (residues 46-676 of the mutant or 57-676 of wild type NADPH-cytochrome P450 reductase) are unable to transfer electrons to cytochrome P450c17 or P4503A4, confirming the importance of the N-terminal sequence for catalysis. Based on the results obtained in the present work, we suggest a scheme of structural topology of the N-terminal hydrophobic sequence of NADPH-cytochrome P450 reductase in the membrane.     

An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.     

Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.     

Subunit location of the iron-sulfur clusters in fumarate reductase from Escherichia coli

The subunit location of the [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in Escherichia coli fumarate reductase has been investigated by EPR studies of whole cells or whole cells extracts of a fumarate reductase deletion mutant with plasmid amplified expression of discrete fumarate reductase subunits or groups of subunits. The results indicate that both the [2Fe-2S] and [3Fe-4S] clusters are located entirely in the iron-sulfur protein subunit. Information concerning the specific cysteine residues that ligate these clusters has been obtained by investigating the EPR characteristics of cells of the deletion mutant amplified with a plasmid coding for the flavoprotein subunit and a truncated iron-sulfur protein subunit. While the results are not definitive with respect to the location of the [4Fe-4S] cluster, they are most readily interpreted in terms of this cluster being entirely in the flavoprotein subunit or bridging between the two catalytic domain subunits. These new results are discussed in light of the amino acid sequences of the two subunits and the sequences of structurally well characterized iron-sulfur proteins containing [2Fe-2S], [3Fe-4S], and [4Fe-4S] centers.     

Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b

The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd-CAB). The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues. The N-terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene. This suggested that the enzyme contained only one species of cytochrome b. Re-evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups. The hydropathy plot of the amino acid sequence predicted five membrane-spanning hydrophobic segments, the first four of which contained a single histidine residue each. These residues could form the axial ligands to the two haem B groups. FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.     

Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution.

p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000. It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller. Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third. An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives. The phases were improved with the B.C. Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis. The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system. The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system. The flavoprotein subunit consists of three domains in close contact. The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site. The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side. The flavin ring lies at the juncture of the first two domains. The third domain lies against the first domain and helps cover the rest of the FAD chain. The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit. The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart. The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.     

Cloning and characterization of sulfite dehydrogenase, two c-type cytochromes, and a flavoprotein of Paracoccus denitrificans GB17: essential role of sulfite dehydrogenase in lithotrophic sulfur oxidation.

A 13-kb genomic region of Paracoccus dentrificans GB17 is involved in lithotrophic thiosulfate oxidation. Adjacent to the previously reported soxB gene (C. Wodara, S. Kostka, M. Egert, D. P. Kelly, and C. G. Friedrich, J. Bacteriol. 176:6188-6191, 1994), 3.7 kb were sequenced. Sequence analysis revealed four additional open reading frames, soxCDEF. soxC coded for a 430-amino-acid polypeptide with an Mr of 47,339 that included a putative signal peptide of 40 amino acids (Mr of 3,599) with a RR motif present in periplasmic proteins with complex redox centers. The mature soxC gene product exhibited high amino acid sequence similarity to the eukaryotic molybdoenzyme sulfite oxidase and to nitrate reductase. We constructed a mutant, GBsoxC delta, carrying an in-frame deletion in soxC which covered a region possibly coding for the molybdenum cofactor binding domain. GBsoxC delta was unable to grow lithoautotrophically with thiosulfate but grew well with nitrate as a nitrogen source or as an electron acceptor. Whole cells and cell extracts of mutant GBsoxC delta contained 10% of the thiosulfate-oxidizing activity of the wild type. Only a marginal rate of sulfite-dependent cytochrome c reduction was observed from cell extracts of mutant GBsoxC delta. These results demonstrated that sulfite dehydrogenase was essential for growth with thiosulfate of P. dentrificans GB17. soxD coded for a periplasmic diheme c-type cytochrome of 384 amino acids (Mr of 39,983) containing a putative signal peptide with an Mr of 2,363. soxE coded for a periplasmic monoheme c-type cytochrome of 236 amino acids (Mr of 25,926) containing a putative signal peptide with an Mr of 1,833. SoxD and SoxE were highly identical to c-type cytochromes of P. denitrificans and other organisms. soxF revealed an incomplete open reading frame coding for a peptide of 247 amino acids with a putative signal peptide (Mr of 2,629). The deduced amino acid sequence of soxF was 47% identical and 70% similar to the sequence of the flavoprotein of flavocytochrome c of Chromatium vinosum, suggesting the involvement of the flavoprotein in thiosulfate oxidation of P. denitrificans GB17.     

Cytochrome c-dependent methacrylate reductase from Geobacter sulfurreducens AM-1.

Geobacter sulfurreducens AM-1 can use methacrylate as a terminal electron acceptor for anaerobic respiration. In this paper, we report on the purification and properties of the periplasmic methacrylate reductase, and show that the enzyme is dependent on the presence of a periplasmic cytochrome c (apparent K(m) = 0.12 microM). The methacrylate reductase was found to be composed of only one polypeptide with an apparent molecular mass of 50 kDa and to contain, bound tightly but not covalently, 1 mol of FAD per mol. The N-terminal amino acid sequence showed sequence similarity to a periplasmic fumarate reductase from Shewanella putrefaciens. However, methacrylate reductase did not catalyze the reduction of fumarate. The periplasmic cytochrome c, which was also purified, had an apparent molecular mass of 30 kDa and contained approximately 4 mol of heme.mol(-1). Cells of G. sulfurreducens AM-1 grown on acetate and methacrylate as an energy source were found to contain all the enzymes required for the oxidation of acetate to CO(2) via the citric acid cycle.     

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.     

Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase

The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114,000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58,000. Recent X-ray crystallographic data from our laboratories [Shamala, N., Lim, L. W., Mathews, F. S., McIntire, W., Singer, T. P., & Hopper, D. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4626-4630], however, indicate an alpha 2 beta 2 structure and a much lower molecular mass (approximately 8000) for the cytochrome subunit. In this paper we report data confirming the conclusions of X-ray crystallographic analysis. From quantitative amino acid analysis, the molecular mass of the flavoprotein monomer is shown to be 48,600 +/- 2200 and that of the cytochrome 8780 +/- 250. These values have been confirmed by gel electrophoresis under denaturing conditions. Gel chromatography under nondenaturing conditions shows that the isolated flavoprotein exists as a dimer, whereas the isolated cytochrome is a monomer. The complete amino acid sequence of the cytochrome c subunit is presented and is shown to have regions of homology to other bacterial c-type cytochromes. The partial N-terminal amino acid sequence (56 amino acids) of the flavoprotein subunit is also reported. The implications of the now established tetrameric structure of the flavocytochrome on data in the literature regarding the redox and association properties of the subunits are examined.     

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.     

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.     

The membrane-bound tetrahaem c-type cytochrome CymA interacts directly with the soluble fumarate reductase in Shewanella

Shewanella spp. demonstrate great variability in the use of terminal electron acceptors in anaerobic respiration; these include nitrate, fumarate, DMSO, trimethylamine oxide, sulphur compounds and metal oxides. These pathways open up possible applications in bioremediation. The wide variety of respiratory substrates for Shewanella is correlated with the evolution of several multi-haem membrane-bound, periplasmic and outer-membrane c-type cytochromes. The 21 kDa c-type cytochrome CymA of the freshwater strain Shewanella oneidensis MR-1 has an N-terminal membrane anchor and a globular tetrahaem periplasmic domain. According to sequence alignments, CymA is a member of the NapC/NirT family. This family of redox proteins is responsible for electron transfer from the quinone pool to periplasmic and outer-membrane-bound reductases. Prior investigations have shown that the absence of CymA results in loss of the ability to respire with Fe(III), fumarate and nitrate, indicating that CymA is involved in electron transfer to several terminal reductases. Here we describe the expression, purification and characterization of a soluble, truncated CymA ('CymA). Potentiometric studies suggest that there are two pairs of haems with potentials of -175 and -261 mV and that 'CymA is an efficient electron donor for the soluble fumarate reductase, flavocytochrome c(3).     

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8-kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined. The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N-terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non-homologous regions which are very rich in alanyl and prolyl residues. The N-terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.