Calculated coupling of electron and proton transfer in the photosynthetic reaction center of Rhodopseudomonas viridis.

Based on new Rhodopseudomonas (Rp.) viridis reaction center (RC) coordinates with a reliable structure of the secondary acceptor quinone (QB) site, a continuum dielectric model and finite difference technique have been used to identify clusters of electrostatically interacting ionizable residues. Twenty-three residues within a distance of 25 A from QB (QB cluster) have been shown to be strongly electrostatically coupled to QB, either directly or indirectly. An analogous cluster of 24 residues is found to interact with QA (QA cluster). Both clusters extend to the cytoplasmic surface in at least two directions. However, the QB cluster differs from the QA cluster in that it has a surplus of acidic residues, more strong electrostatic interactions, is less solvated, and experiences a strong positive electrostatic field arising from the polypeptide backbone. Consequently, upon reduction of QA or QB, it is the QB cluster, and not the QA cluster, which is responsible for substoichiometric proton uptake at neutral pH. The bulk of the changes in the QB cluster are calculated to be due to the protonation of a tightly coupled cluster of the three Glu residues (L212, H177, and M234) within the QB cluster. If the lifetime of the doubly reduced state QB2- is long enough, Asp M43 and Ser L223 are predicted to also become protonated. The calculated complex titration behavior of the strongly interacting residues of the QB cluster and the resulting electrostatic response to electron transfer may be a common feature in proton-transferring membrane protein complexes.     

Analysis of correlated mutations in HIV-1 protease using spectral clustering

MOTIVATION: The ability of human immunodeficiency virus-1 (HIV-1) protease to develop mutations that confer multi-drug resistance (MDR) has been a major obstacle in designing rational therapies against HIV. Resistance is usually imparted by a cooperative mechanism that can be elucidated by a covariance analysis of sequence data. Identification of such correlated substitutions of amino acids may be obscured by evolutionary noise. RESULTS: HIV-1 protease sequences from patients subjected to different specific treatments (set 1), and from untreated patients (set 2) were subjected to sequence covariance analysis by evaluating the mutual information (MI) between all residue pairs. Spectral clustering of the resulting covariance matrices disclosed two distinctive clusters of correlated residues: the first, observed in set 1 but absent in set 2, contained residues involved in MDR acquisition; and the second, included those residues differentiated in the various HIV-1 protease subtypes, shortly referred to as the phylogenetic cluster. The MDR cluster occupies sites close to the central symmetry axis of the enzyme, which overlap with the global hinge region identified from coarse-grained normal-mode analysis of the enzyme structure. The phylogenetic cluster, on the other hand, occupies solvent-exposed and highly mobile regions. This study demonstrates (i) the possibility of distinguishing between the correlated substitutions resulting from neutral mutations and those induced by MDR upon appropriate clustering analysis of sequence covariance data and (ii) a connection between global dynamics and functional substitution of amino acids.     

Partial characterization of a novel cathepsin L-like protease from Fasciola hepatica

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CH&dbond;CH(2)-CO(2)R (where R = Me/Et/Bu(t)). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.     

Spatial sign-alternating charge clusters in globular proteins

Large sign-alternating charge clusters formed by the charged side groups of amino acid residues and N- and C-terminal groups were found in the majority of considered globular proteins, namely 235 in a total of 274 protein structures, i.e. 85.8%. The clusters were determined by the criteria proposed earlier: charged groups were included in the cluster if their charged N and O atoms were located at distances between 2.4 and 7.0 A. The set of selected proteins consisted of known non-homologous protein structures from the Protein Data Bank with a resolution less than or equal to 2.5 A and pair sequence similarity less than 25%. Molecular masses of the proteins were from 5.5 to 91.5 kDa and protein chain length from 50 to 830 residues. The distribution of charged groups on the protein surface between isolated charged groups, small clusters with two and three groups, and large clusters with four or more groups were found to be approximately similar making 33, 35 and 32% of the total amount of protein charged groups, respectively. The large sign-alternating charge clusters with four or more charged groups were studied in greater detail. The amount of such clusters depends on the protein chain length. The small proteins contain 1-3 clusters while the large proteins display 4-6 or more clusters. On average, 1.5 clusters per each 100 residues were observed. In contrast with this, the size of a cluster, i.e. the number of charged groups inside a cluster, does not depend on the protein molecular mass, and large clusters are observed for proteins from a range of molecular masses. Clusters consisting of four to six charged groups occur most frequently, although extra large clusters are also often revealed. We can conclude that sign-alternating charge clusters are a common feature of the protein surface of globular protein. They are suggested to play a general functional role as a local polar factor of protein surface.     

Effect of spatial coupling of amino acid residues on the possibility of mutational substitution in globular proteins

Amino acid residues can be divided into similar groups by frequencies of interreplacements in the evolutionary pathway and by trends to spatial contacts at the tertiary structures of globular proteins. Each residue was compared to the cluster of spatial surrounding--the totality of residues spacially drawn together. 5210 clusters in 32 unhomologous proteins with established tertiary structure and 6447 clusters formed only by variables amino acid residues were analysed. Spatial contacts among residues were studied depending on the secondary structure and the amount of residues in a cluster. It was assumed that functionally admissible mutations may be defined, first of all, by the degree of neighboring of amino acid residues in the spatial surrounding.     

Locations of methyl groups in 28 S rRNA of Xenopus laevis and man. Clustering in the conserved core of molecule

28 S ribosomal RNA from several vertebrate species contains some 68 to 70 methyl groups. Evidence described in this paper enables some 58 methyl groups to be located in the primary structure of 28 S ribosomal RNA from Xenopus laevis. Most of the locations are unambiguous but a few are currently tentative. In human 28 S ribosomal RNA the great majority of the same sites are methylated as in Xenopus, but there are a few differences between the respective methyl group distributions. The main features of the methyl group distribution are as follows. (1) All of the identified methyl groups are in conserved core regions of 28 S ribosomal RNA. (2) Methylation is much more heavily concentrated in the 3' region of the molecule than in the 5' region (in contrast to 18 S ribosomal RNA, in which there is a major cluster of 2'-O-methyl groups in the 5' region). (3) In addition to the heavily methylated 3' region, clusters of methyl groups occur elsewhere in 28 S ribosomal RNA in the vicinity of domain boundaries. For domains 3 to 6, the two ends of each domain are united in a helix and are linked to adjacent domains either directly or by short single-stranded regions. It therefore follows that the methyl groups near the boundaries of these domains come together into the same general region of the three-dimensional structure. Within this large-scale pattern of distribution, methyl groups occur in a variety of local environments, examples of which are discussed. The triply methylated sequence Am-Gm-Cm-A occurs in a short single-stranded region which links domain 3 to domain 4. Near the 3' end of domain 5 there is a cluster of 11 methyl groups including a 2'-O-methyl pseudouridine in a tract of 160 nucleotides whose sequence is totally conserved between Xenopus and man. These methyl groups are variously distributed between single-stranded regions and short or imperfect but conserved helices. A further cluster of methyl groups including the highly conserved Um-Gm-psi sequence occurs in a region of domain 6 which is implicated in peptidyl transfer. Possible relationships between methylation and other events in ribosome maturation are discussed.     

Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly

The O-antigen is an important component of the outer membrane of Gram-negative bacteria. It is a repeat unit polysaccharide and consists of a number of repeats of an oligosaccharide, the O-unit, which generally has between two and six sugar residues. O-Antigens are extremely variable, the variation lying in the nature, order and linkage of the different sugars within the polysaccharide. The genes involved in O-antigen biosynthesis are generally found on the chromosome as an O-antigen gene cluster, and the structural variation of O-antigens is mirrored by genetic variation seen in these clusters. The genes within the cluster fall into three major groups. The first group is involved in nucleotide sugar biosynthesis. These genes are often found together in the cluster and have a high level of identity. The genes coding for a significant number of nucleotide sugar biosynthesis pathways have been identified and these pathways seem to be conserved in different O-antigen clusters and across a wide range of species. The second group, the glycosyl transferases, is involved in sugar transfer. They are often dispersed throughout the cluster and have low levels of similarity. The third group is the O-antigen processing genes. This review is a summary of the current knowledge on these three groups of genes that comprise the O-antigen gene clusters, focusing on the most extensively studied E. coli and S. enterica gene clusters.     

Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils

Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.     

Stabilizing interactions in the dimer interface of alpha-subunit in Escherichia coli RNA polymerase: a graph spectral and point mutation study

The formation of alpha(2) dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the alpha-subunit dimerizes through its N-terminal domain (NTD). The crystal structures of the alpha-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the alpha-NTD of E. coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E. coli alpha-NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core. The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Residues 35F and 46I were predicted to be important in the stability of the alpha-dimer interface, with 35F forming the center of the cluster. The predictions were tested by isolating single-point mutants alpha-F35A and alpha-I46S on the dimer interface, which were found to disrupt dimerization. Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the alpha-NTD.     

Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease-including 22 not previously associated with drug resistance-were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 A of each other-many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.     

Sites of methyl esterification and deamination on the aspartate receptor involved in chemotaxis

The receptors involved in bacterial chemotaxis are post-translationally modified by specific enzymes which catalyze the deamination of glutaminyl residues and the methyl esterification and demethylation of glutamyl residues. In this work we identify the sites of these covalent modifications on the aspartate receptor from Salmonella typhimurium. These were identified using the properties of the Staphylococcus aureus V8 protease which cleaves peptide bonds following glutamyl but not glutaminyl residues. We show here that bonds following methyl-esterified glutamyl residues are also resistant to the protease. A comparison of the fragments obtained after V8 protease cleavage of methyl-esterified (or deaminated) peptides with the fragments from the corresponding unmodified peptides immediately yields the sites of modification. Three of the four methyl-esterified glutamyl residues are located near the middle of the receptor amino acid sequence; one of these is synthesized as a glutaminyl residue and is deaminated by the esterase to form a glutamyl residue. The fourth site of methyl esterification is located near the carboxyl terminus. All four sites occupy analogous positions in a well-conserved arrangement of residues which may form a binding site for the esterase and the methyltransferase.     

Hydrophobic clusters in protein structures

Globular proteins fold such that the hydrophobic groups are packed inside forming hydrophobic clusters, and the hydrophilic groups are present on the surface. In this article we analyze clusters of hydrophobic groups of atoms in 781 protein structures selected from the PDB. Our analysis showed that every structure consists of two types of clusters: at least one large cluster that forms the hydrophobic core and probably dictates the protein fold; and numerous smaller clusters, which might be involved in the stabilization of the fold. We also analyzed the preference of the hydrophobic groups in each of the amino acids toward forming hydrophobic clusters. We find that hydrophobic groups from the hydrophilic amino acids also contribute toward cluster formation. Proteins 2008. © 2008 Wiley‐Liss, Inc.     

Demonstration of the importance and usefulness of manipulating non-active-site residues in protein design

Do non-active-site residues participate in protein function in a more direct way than just by holding the static framework of the protein molecule? If so, how important are they? As a model to answer these questions, ATB17, which is a mutant of aspartate aminotransferase created by directed evolution, is an ideal system because it shows a 10(6)-fold increase in the catalytic efficiency for valine but most of its 17 mutated residues are non-active-site residues. To analyze the roles of the mutations in the altered function, we divided the mutations into four groups, namely, three clusters and the remainder, based on their locations in the three-dimensional structure. Mutants with various combinations of the clusters were constructed and analyzed, and the data were interpreted in the context of the structure-function relationship of this enzyme. Each cluster shows characteristic effects: for example, one cluster appears to enhance the catalytic efficiency by fixing the conformation of the enzyme to that of the substrate-bound form. The effects of the clusters are largely additive and independent of each other. The present results illustrate how a protein function is dramatically modified by the accumulation of many seemingly inert mutations of non-active-site residues.     

Identification and expressional analysis of two cathepsins from half-smooth tongue sole (Cynoglossus semilaevis)

Cathepsins are a family of lysosomal proteases that play an important role in protein degradation, antigen presentation, apoptosis, and inflammation. Cathepsins are divided into three groups, i.e., cysteine protease, serine protease, and aspartic protease. Cathepsin D and cathepsin L, which are aspartic protease and cysteine protease respectively, have been identified in a number of teleosts; however, the immunological relevance of fish cathepsins is largely unknown. In this study, we cloned and analyzed the expression profiles of a cathepsin D (CsCatD) and a cathepsin L (CsCatL) homologs from half-smooth tongue sole (Cynoglossus semilaevis). CsCatD is composed of 396 amino acid residues and shares 67.6-88.4% overall sequence identities with fish and human cathepsin D. Structurally CsCatD possesses an aspartic endopeptidase domain, which contains two conserved aspartic acid residues that form the catalytic site. CsCatL is 336 residues in length and shares 64.7-90.2% overall sequence identities with fish and human cathepsin L. CsCatL has an N-terminal cathepsin propeptide inhibitor domain followed by a Papain family cysteine protease domain, the latter containing four conserved catalytic residues: Gln-133, Cys-139, His-279, and Asn-303. Recombinant CsCatL purified from Escherichia coli exhibited apparent protease activity. Quantitative real time RT-PCR analysis detected constitutive expression of CsCatD and CsCatL in multiple tissues, with the lowest level found in heart and the highest level found in liver. Experimental challenge of tongue sole with the bacterial pathogen Vibrio anguillarum and megalocytivirus caused significant inductions of both CsCatD and CsCatL expression in kidney and spleen in time-dependent manners. Immunization of the fish with a subunit vaccine also enhanced CsCatD and CsCatL expression in the first week post-vaccination. These results suggest involvement of CsCatD and CsCatL in host immune reactions to bacterial and viral infections and in the process of antigen-induced immune response.     

Theoretical investigation of the behavior of titratable groups in proteins.

This paper presents a theoretical analysis of the titration behavior of strongly interacting titratable residues in proteins. Strongly interacting titratable residues exist in many proteins such as for instance bacteriorhodopsin, cytochrome c oxidase, cytochrome bc(1), or the photosynthetic reaction center. Strong interaction between titratable groups can lead to irregular titration behavior. We analyze under which circumstances titration curves can become irregular. We demonstrate that conformational flexibility alone can not lead to irregular titration behavior. Strong interaction between titratable groups is a necessary, but not sufficient condition for irregular titration curves. In addition, the two interacting groups also need to titrate in the same pH-range. These two conditions together lead to irregular titration curves. The mutation of a single residue within a cluster of interacting titratable residues can influence the titration behavior of the other titratable residues in the cluster. We demonstrate this effect on a cluster of four interacting residues. This example underlines that mutational studies directed at identifying the role of a certain titratable residue in a cluster of interacting residues should always be accompanied by an analysis of the effect of the mutation on the titration behavior of the other residues.     

Genomic organization and functional expression of differentially regulated cysteine protease genes of Leishmania donovani complex

For the first time, we report the genomic organization and characterization of Cathepsin L-like cysteine protease gene cluster from the members of Leishmania donovani complex. The cysteine protease gene cluster of Leishmania chagasi has five copies of tandemly arranged genes. The first gene (Ldccys1A) is identical to Ldccys1 cDNA and is predominantly expressed in promastigotes. The last gene (Ldccys1E) is identical to Ldccys1A with a 13 amino acids deletion in the mature domain, including one of the active site histidine residues and a truncated carboxyl terminal extension. It has a diverged 3' untranslated region and is also constitutively expressed in the parasite. Results from rapid amplification of cDNA ends (RACE) suggest that there are three different types of 3' untranslated regions, one of them is identical to that of Ldccys1A whereas another to Ldccys1E. The third one is also identical to Ldccys1A, but has a 154 nucleotides deletion near the polyA region and this gene is constitutively expressed. Gene organization and expression in L. donovani cluster is similar to that of L. chagasi. However, the last gene (Lddcys1F) is different from Ldccys1E as it lacks 13 amino acid deletions. Also, L. donovani possesses an additional copy of the gene (Lddcys1E), which is located away from the cluster. Furthermore, for the first time we have expressed full-length cysteine protease genes in an insect expression system. Ldccys1A and Ldccys1F cleaved gelatin whereas Ldccys1E was found to be inactive in gelatin assays.     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.     

Additivity in both thermodynamic stability and thermal transition temperature for rubredoxin chimeras via hybrid native partitioning

Given any operational definition of pairwise interaction, the set of residues that differ between two structurally homologous proteins can be uniquely partitioned into subsets of clusters for which no such interactions occur between clusters. Although hybrid protein sequences that preserve such clustering are consistent with tertiary structures composed of only parental native-like interactions, the stability of such predicted structures will depend upon the physical robustness of the assumed interaction potential. A simple distance cutoff criterion was applied to the most thermostable protein known to predict such a seven-residue cluster in the metal binding site region of Pyrococcus furiosus rubredoxin and a mesophile homolog. Both conformational stability and thermal transition temperature measurements demonstrate that 39% of the differential stability arises from these seven residues.     

The amino acid sequences of two 13 kDa polypeptides and partial amino acid sequence of 30 kDa polypeptide of complex I from bovine heart mitochondria: possible location of iron-sulfur clusters

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of iron-sulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10,536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13,130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.     

Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase.

Trimethylamine dehydrogenases from bacterium W3A1 and Hyphomicrobium X and the dimethylamine dehydrogenase from Hyphomicrobium X were found to contain only one kind of subunit. The millimolar absorption coefficient of a single [4Fe-4S] cluster in trimethylamine dehydrogenase from bacterium W3A1 was estimated to be 14.8 mM-1 . cm-1 at 443 nm. From this value a 1:1 stoicheiometry of the prosthetic groups, 6-S-cysteinyl-FMN and the [4Fe-4S] cluster, was established. Millimolar absorption coefficients of the three enzymes were in the range 49.4-58.7 mM-1 . cm-1 at approx. 440 nm. This range of values is consistent with the presence of two [4Fe-4S] clusters and two flavin residues, for which the millimolar absorption coefficient had earlier been found to be 12.3 mM-1 . cm-1 at 437 nm. The N-terminal amino acid was alanine in each of the three enzymes. Sequence analysis of the first 15 residues from the N-terminus of dimethylamine dehydrogenase indicated a single unique sequence. Two identical subunits, each containing covalently bound 6-S-cysteinyl-FMN and a [4Fe-4S] cluster, in each of the enzymes are therefore indicated.