C-type Heme Research Articles

The small domain of cytochromeffrom the psychrophileChlamydomonas raudensisUWO 241 modulates the apparent molecular mass and decreases the accumulation of cytochromefin the mesophileChlamydomonas reinhardtii

Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e-/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments.

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately -500 mV (vs. the standard hydrogen electrode). Across this potential window the UV-vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.

The Crystal Structure of Cytochrome P460 ofNitrosomonas europaeaReveals 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.

Crystal Structure of the 16 Heme Cytochrome from Desulfovibrio gigas: A Glycosylated Protein in a Sulphate-reducing Bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 Å resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis

In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-β- d-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd1Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

A M106H variant, where M106 is a c-type heme iron axial ligand, of cytochrome cd(1) nitrite reductase is an inactive protein in vivo. Expression of the holoprotein in Paracoccus pantotrophus required generation of nitric oxide during cell growth through simultaneous expression of an exogenous copper nitrite reductase from Alcaligenes faecalis. In the absence of the latter protein, only a semi-apo form of M106H cytochrome cd(1) was formed. Thus it was demonstrated that expression of the chromosomal nir genes for d(1) heme biosynthesis in P. pantotrophus is NO-dependent, probably mediated by the transcription factor NNR, and a route to low or zero activity mutants had been established. The value of such variants for mechanistic studies on cytochrome cd(1) is illustrated by the use of M106H to demonstrate that the d(1) heme potential can be resolved and measured at approximately +175 mV with the c heme shifted to -60 mV, consistent with its bishistidinyl coordination. The unusual highly cooperative and strongly hysteretic redox titration of the wild type is lost in the M106H protein. The same c heme midpoint potential was observed in a M106H variant of a c-domain construct. The difference between d(1) heme and c heme redox potentials has allowed preparation of a M106H protein with oxidized c heme and reduced d(1) heme. This one electron reduced form will reduce nitrite to nitric oxide, but the latter remains bound to the resulting fully oxidized enzyme.

Redox infrared markers of the heme and axial ligands in microperoxidase: bases for the analysis of c-type cytochromes

Structural changes accompanying the change in the redox state of microperoxidase-8 (MP8), the heme-octapeptide obtained from cytochrome c, and its complexes with (methyl)imidazole ligands were studied by electrochemically induced Fourier transform IR (FTIR) difference spectroscopy. To correlate with confidence IR modes with a specific electronic state of the iron, we used UV-vis and electron paramagnetic resonance spectroscopy to define precisely the heme spin state in the samples at the millimolar concentration of MP8 required for FTIR difference spectroscopy. We identified four intense redox-sensitive IR heme markers, nu38 at 1,569 cm(-1) (ox)/1,554 cm(-1) (red), nu42 at 1,264 cm(-1) (ox)/1,242 cm(-1) (red), nu43 at 1,146 cm(-1) (ox), and nu44 at 1,124-1,128 cm(-1) (ox). The intensity of nu42 and nu43 was clearly enhanced for low-spin imidazole-MP8 complexes, while that of nu44 increased for high-spin MP8. These modes can thus be used as IR markers of the iron spin state in MP8 and related c-type cytochromes. Moreover, one redox-sensitive band at 1,044 cm(-1) (red) is attributed to an IR marker specific of c-type hemes, possibly the delta(CbH3)(2,4) heme mode. Other redox-sensitive IR bands were assigned to the MP8 peptide backbone and to the fifth and sixth axial heme ligands. The distinct IR frequencies for imidazole (1,075 cm(-1)) and histidine (1,105 cm(-1)) side chains in the imidazole-MP8 complex allowed us to provide the first direct determination of their pKa at pH 9 and 12, respectively.

GHP, a new c‐type green heme protein from Halochromatium salexigens and other proteobacteria

We have isolated a minor soluble green-colored heme protein (GHP) from the purple sulfur bacterium, Halochromatium salexigens, which contains a c-type heme. A similar protein has also been observed in the purple bacteria Allochromatium vinosum and Rhodopseudomonas cryptolactis. This protein has wavelength maxima at 355, 420, and 540 nm and remains unchanged upon addition of sodium dithionite or potassium ferricyanide, indicating either an unusually low or high redox potential, respectively. The amino-acid sequence indicates one heme per peptide chain of 72 residues and reveals weak similarity to the class I cytochromes. The usual sixth heme ligand methionine in these proteins appears to be replaced by a cysteine in GHP. Only one known cytochrome has a cysteine sixth ligand, SoxA (cytochrome c-551) from thiosulfate-oxidizing bacteria, which is low-spin and has a high redox potential because of an un-ionized ligand. The native size of GHP is 34 kDa, its subunit size is 11 kDa, and the net charge is -12, accounting for its very acidic nature. A database search of complete genome sequences reveals six homologs, all hypothetical proteins, from Oceanospirillum sp., Magnetococcus sp., Thiobacillus denitrificans, Dechloromonas aromatica, Thiomicrospira crunogena and Methylobium petroleophilum, with sequence identities of 35-64%. The genetic context is different for each species, although the gene for GHP is transcriptionally linked to several other genes in three out of the six species. These genes, coding for an RNAse, a protease/chaperone, a GTPase, and pterin-4a-carbinolamine dehydratase, appear to be functionally related to stress response and are linked in at least 10 species.

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

Characterization of ac-type heme-containing PAS sensor domain fromGeobacter sulfurreducensrepresenting a novel family of periplasmic sensors inGeobacteraceaeand other bacteria

Geobacter sulfurreducens encodes one of the largest numbers of proteins annotated as parts of the two-component signal transduction and/or chemotaxis pathways. Ten of these signal transducers have homologous periplasmic sensor domains that contain the sequence signature for c-type hemes. One such sensor domain encoded by gene GSU0303 was isolated and characterized. The protein was expressed in Escherichia coli and was isolated as two colored species (green and red). The green species is a monomer of the sensor domain with a five-coordinated high-spin heme and the red species is probably a noncovalent dimer of the sensor domain which might have an uncharacterized ligand bound to the dimer. The UV-VIS spectrum of the green species indicates that it has a c'-type heme, but its structure is predicted to be homologous to CitA, a periplasmic PAS domain that does not contain heme. The GSU0303 sensor domain represents a previously unreported family of PAS-type periplasmic sensor domains that contain c-type hemes; these proteins could be part of an important mechanism for sensing redox potential or small ligands in the periplasm. Homologs to the sensor domains we identified in G. sulfurreducens are observed in various bacteria although they occur in larger numbers in the Geobacteraceae.

NO Reductase Activity of the Tetraheme Cytochromec554ofNitrosomonas europaea

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is believed to function as an electron-transfer protein from hydroxylamine oxidoreductase (HAO). We show here that cyt c(554) also has significant NO reductase activity. The protein contains one high-spin and three low-spin c-type hemes. HAO catalyzed reduction of the cyt c(554), ligand binding, intermolecular electron transfer, and kinetics of NO reduction by cyt c(554) have been investigated. We detect the formation of a NO-bound ferrous heme species in cyt c(554) by EPR and Mössbauer spectroscopies during the HAO catalyzed oxidation of hydroxylamine, indicating that N-oxide intermediates produced from HAO readily bind to cyt c(554). In the half-reduced state of cyt c(554), we detect a spin interaction between the [FeNO](7) state of heme 2 and the low-spin ferric state of heme 4. We find that ferrous cyt c(554) will reduce NO at a rate greater than 16 s(-1), which is comparable to rates of other known NO reductases. Carbon monoxide or nitrite are shown not to bind to the reduced protein, and previous results indicate the reactions with O(2) are slow and that a variety of ligands will not bind in the oxidized state. Thus, the enzymatic site is highly selective for NO. The NO reductase activity of cyt c(554) may be important during ammonia oxidation in N. europaea at low oxygen concentrations to detoxify NO produced by reduction of nitrite or incomplete oxidation of hydroxylamine.

Molecular and catalytic properties of a novel cytochrome c nitrite reductase from nitrate-reducing haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

A highly active cytochrome c nitrite reductase from the haloalkaliphilic sulfur-oxidizing non-ammonifying bacterium Tv. nitratireducens strain ALEN 2 (TvNiR) was isolated and purified to apparent electrophoretic homogeneity. The enzyme catalyzes reductive conversion of nitrite and hydroxylamine to ammonia without release of any intermediates, as well as reduction of sulfite to sulfide. TvNiR also possesses peroxidase activity. In solution TvNiR exists as a stable hexamer with molecular mass of about 360 kDa. Each TvNiR subunit with molecular mass of 64 kDa contains, as defined from spectral properties and sequence analysis, eight c-type haems. Seven of them are coordinated by the characteristic CXXCH motifs for haem c binding, while one is bonded by the unique CXXCK motif. So far, this motif coordinating the catalytic haem was found only in bacterial cytochrome c nitrite reductases (ccNiRs). All the residues essential for catalysis in the known ccNiRs were also identified in TvNiR. However, TvNiR is only distantly related to known bacterial ammonifying dissimilatory ccNiRs, sharing no more than 20% homology.

Evidence for Redox Cooperativity betweenc-Type Hemes of MauG Which Is Likely Coupled to Oxygen Activation during Tryptophan Tryptophylquinone Biosynthesis

MauG is a novel 42 kDa diheme protein which is required for the biosynthesis of tryptophan tryptophylquinone, the prosthetic group of methylamine dehydrogenase. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes and the overall redox properties of MauG are described. The absorption maxima for the Soret peaks of the oxidized and reduced hemes are 403 and 418 nm for the low-spin heme and 389 and 427 nm for the high-spin heme, respectively. The resonance Raman spectrum of oxidized MauG exhibits a set of marker bands at 1503 and 1588 cm(-1) which exhibit frequencies similar to those of the nu3 and nu2 bands of c-type heme proteins with bis-histidine coordination. Another set of marker bands at 1478 and 1570 cm(-1) is characteristic of a high-spin heme. Two distinct oxidation-reduction midpoint potential (E(m)) values of -159 and -244 mV are obtained from spectrochemical titration of MauG. However, the two nu3 bands located at 1478 and 1503 cm(-1) shift together to 1467 and 1492 cm(-1), respectively, upon reduction, as do the Soret peaks of the low- and high-spin hemes in the absorption spectrum. Thus, the two hemes with distinct spectral properties are reduced and oxidized to approximately the same extent during redox titrations. This indicates that the high- and low-spin hemes have similar intrinsic E(m) values but exhibit negative redox cooperativity. After the first one-electron reduction of MauG, the electron equilibrates between hemes. This makes the second one-electron reduction of MauG more difficult. Thus, the two E(m) values do not describe redox properties of distinct hemes, but the first and second one-electron reductions of a diheme system with two equivalent hemes. The structural and mechanistic implications of these findings are discussed.

Biophysical Properties of a c-Type Heme in Chemotaxis Signal Transducer Protein DcrA

Chemotaxis signal transducer protein DcrA from a sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was previously shown to contain a c-type heme in its periplasmic domain (DcrA-N) for sensing redox and/or oxygen [Fu et al. (1994) J. Bacteriol. 176, 344-350], which is the first example of a heme-based sensor protein containing a c-type heme as a prosthetic group. Optical absorption and resonance Raman spectroscopies indicates that heme c in DcrA-N shows a redox-dependent ligand exchange. Upon reduction, a water molecule that may be the sixth ligand of the ferric heme c is replaced by an endogenous amino acid. Although the reduced heme in DcrA-N is six-coordinated with two endogenous axial ligands, CO can easily bind to the reduced heme to form CO-bound DcrA-N. Reaction of the reduced DcrA-N with molecular oxygen results in autoxidation to form a ferric state without forming any stable oxygen-bound form probably due to the extremely low redox potential of DcrA-N (-250 mV). Our study supports the initial idea by Fu et al. that DcrA would act as a redox and/or oxygen sensor, in which the ligand exchange between water and an endogenous amino acid is a trigger for signal transduction. While the affinity of CO to DcrA-N (Kd = 138 microM) is significantly weak compared to those of other heme proteins, we suggest that CO might be another physiological effector molecule.

A new soluble 10 kDa monoheme cytochrome c-552 from the anammox bacterium Candidatus “ Kuenenia stuttgartiensis”

The chemolithoautotrophic anammox bacterium Candidatus “ Kuenenia stuttgartiensis” grows anaerobically using ammonium as electron donor for nitrite reduction. More than 10% of the proteins in cell extracts of “ K. stuttgartiensis” consist of c-type heme proteins. A 10 kDa soluble cytochrome c was purified from cell extracts using ultracentrifugation and anion exchange chromatography. The UV/Vis spectrum of the reduced cytochrome showed the γ, β and α absorption maxima at 419, 522 and 552 nm, respectively. The N-terminal amino acid sequence and peptide fragments of the tryptic digest of the protein were used to identify the corresponding gene. Analysis of the gene product showed that the protein was preceded by a 30 amino acids long leader sequence and that it belonged to the low-spin class ID cytochrome c. The CXXCH motive was located at the N-terminal site of the protein. The gene organization of the cytochrome showed some resemblance to cytochrome c clusters of unknown function in the genome of Nitrosomonas europaea and Geobacter sulfurreducens PCA.

1H NMR studies of paramagnetic ferricytochrome c-551 from Pseudomonas aeruginosa at high pH: The role of histidine 16 in the spin transition

Cytochrome c-551 from the mesophile Pseudomonas aeruginosa is an electronic transfer protein that contains 82 amino-acid residues and a c-type heme as the prosthetic group with low spin Fe(II) in the reduced form and low spin Fe(III) in the oxidized form of cytochrome c-551. We have studied the electronic properties of ferricytochrome c-551 from P. aeruginosa at high pH (9–11.4) by means of paramagnetic 1H NMR spectra and the T 1 and T 2 values of isotropically shifted proton resonances. We have also analyzed the temperature dependence of the hyperfine-shifts. Resonance assignment of some signals was based on 2D saturation transfer experiments, EXSY. These results indicate the existence of high-spin iron(III) cytochrome c-551 ( S = 5/2, 6A 1) with penta or hexacoordinated symmetry at high pH. A spin transition from low-spin iron(III) to high-spin iron(III) has been produced. This transition can be associated with the deprotonation of the axially coordinated amino-acid to iron, His-16. We also discuss the spin crossover between different ferricytochromes c.

Molecular Basis of Intramolecular Electron Transfer in Sulfite-oxidizing Enzymes Is Revealed by High Resolution Structure of a Heterodimeric Complex of the Catalytic Molybdopterin Subunit and a c-Type Cytochrome Subunit

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 A) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.

Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures

Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.

MauG-Dependent in Vitro Biosynthesis of Tryptophan Tryptophylquinone in Methylamine Dehydrogenase

Tryptophan tryptophylquinone (TTQ) is the prosthetic group of methylamine dehydrogenase (MADH) and is synthesized through post-translational modification of two endogenous tryptophan residues. This modification involves two oxygenation reactions and one cross-linking reaction. It is clearly shown that the incorporation of the second oxygen into betaTrp57 and the covalent cross-linking of betaTrp57 to betaTrp108 are MauG-dependent processes. These reaction steps are severely compromised in vivo when mauG is mutated or deleted. These steps may then be catalyzed in vitro upon addition of MauG to the isolated biosynthetic intermediates. These results also show that TTQ formation is linked to proper assembly of subunits during MADH biosynthesis. Last, these results demonstrate a novel function for the c-type heme protein, MauG, which is consistent with its atypical physical properties. These results are the first description of an enzyme-mediated biosynthesis of a protein-derived cofactor in vitro.

Active Site Aspartate Residues Are Critical for Tryptophan Tryptophylquinone Biogenesis in Methylamine Dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue betaTrp57 and covalent cross-linking of betaTrp57 to betaTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues betaAsp76 and betaAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative betaD76N and betaD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. betaD76N MADH possessed the six disulfides but had no oxygen incorporated into betaTrp57 and was completely inactive. The betaD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the betaD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.