DNA-[adenine] methylation in lower eukaryotes.

DNA methylation in lower eukaryotes, in contrast to vertebrates, can involve modification of adenine to N6-methyladenine (m6A). While DNA-[cytosine] methylation in higher eukaryotes has been implicated in many important cellular processes, the function(s) of DNA-[adenine] methylation in lower eukaryotes remains unknown. I have chosen to study the ciliate Tetrahymena thermophila as a model system, since this organism is known to contain m6A, but not m5C, in its macronuclear DNA. A BLAST analysis revealed an open reading frame (ORF) that appears to encode for the Tetrahymena DNA-[adenine] methyltransferase (MTase), based on the presence of motifs characteristic of the enzymes in prokaryotes. Possible biological roles for DNA-[adenine] methylation in Tetrahymena are discussed. Experiments to test these hypotheses have begun with the cloning of the gene. Orthologous ORFs are also present in three species of the malarial parasite Plasmodium. They are compared to one another and to the putative Tetrahymena DNA-[adenine] MTase. The gene from the human parasite P. falciparum has been cloned.

[Molecular enzymology of phage T4 Dam DNA-methyltransferase]. / Moleculiarnaia énzymologiia Dam-DNK-metiltranferazy faga T4.

The review reflects results of studies on the molecular mechanism of phage T4 Dam DNA-methyltransferase action. The enzyme (T4Dam) catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to N6-adenine position in the palindromic recognition sequence GATC (EC 2.1.1.72). The enzyme subunit structure, substrate-binding and kinetic parameters for a wide range of native and modified oligonucleotide duplexes, as well as steady-state reaction kinetic scheme, included T4Dam isomerization to catalytically active form, are considered. The found mechanisms of DNA induced T4Dam dimerization, target base flipping, enzyme reorientation in an asymmetrically modified recognition sequence, effector action of reaction substrates and processive methylation of DNA substrates, containing more than one specific site, are discussed. The results obtained with T4Dam may be useful for understanding mechanisms of action of other homologous enzymes, most of all for specimens of numerous family of Dam DNA-methyltransferases.

A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase.

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.

Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage.

Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.

Intrinsic DNA distortion of the bacteriophage Mu momP1 promoter is a negative regulator of its transcription. A novel mode of regulation of toxic gene expression.

The momP1 promoter of the bacteriophage Mu mom operon is an example of a weak promoter. It contains a 19-base pair suboptimal spacer between the -35 (ACCACA) and -10 (TAGAAT) hexamers. Escherichia coli RNA polymerase is unable to bind to momP1 on its own. DNA distortion caused by the presence of a run of six T nucleotides overlapping the 5' end of the -10 element might prevent RNA polymerase from binding to momP1. To investigate the influence of the T(6) run on momP1 expression, defined substitution mutations were introduced by site-directed mutagenesis. In vitro probing experiments with copper phenanthroline ((OP)(2)Cu) and DNase I revealed distinct differences in cleavage patterns among the various mutants; in addition, compared with the wild type, the mutants showed an increase (variable) in momP1 promoter activity in vivo. Promoter strength analyses were in agreement with the ability of these mutants to form open complexes as well as to produce momP1-specific transcripts. No significant role is attributed to the overlapping and divergently organized promoter, momP2, in the expression of momP1 activity, as determined by promoter disruption analysis. These data support the view that an intrinsic DNA distortion in the spacer region of momP1 acts in cis as a negative element in mom operon transcription. This is a novel mechanism of regulation of toxic gene expression.

Pre-steady state kinetics of bacteriophage T4 dam DNA-[N(6)-adenine] methyltransferase: interaction with native (GATC) or modified sites.

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S:-adenosyl-L-methionine (AdoMet) to the N(6)-position of adenine [generating N(6)-methyladenine and S:-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.

Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase.

The interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with 24mer synthetic oligonucleotide duplexes having different purine base substitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays. The substitutions were introduced in either the upper or lower strand: guanine by 7-deazaguanine (G-->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-aminopurine (A-->N). The effects of each base modification on binding/methylation were approximately equivalent for both strands. G-->D and G-->N substitutions resulted in a sharp decrease in binary complex formation. This suggests that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex. In contrast, A-->P and A-->N substitutions were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the central A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [3H]methylated product. In all cases the substrate dependencies of bursts and methylation rates were proportional to each other. For the perfect 24mer k cat = 0.014/s and K m = 7.7 nM was obtained. In contrast to binary complex formation the two guanine substitutions exerted relatively minor effects on catalytic turnover (the k cat was reduced at most 2. 5-fold), while the two adenine substitutions showed stronger effects (5- to 15-fold reduction in k cat). The effects of base analog substitutions on K m(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).

Unusual transcriptional and translational regulation of the bacteriophage Mu mom operon.

The bacteriophage Mu mom gene encodes a novel DNA modification that protects the viral genome against a wide variety of restriction endonucleases. Expression of mom is subject to a series of unusual regulatory controls. Transcription requires the action of a phage-encoded protein, C, which binds (probably as a dimer) the mom promoter from -33 to -52 (with respect to the transcription start site) in two adjacent DNA major grooves on one face of the helix. No apparent direct interaction between C and the host RNA polymerase (RNAP) is evident; however, C binding alters mom DNA conformation. In the absence of C, RNAP binds the mom promoter at a site that results in transcription in a direction away from the mom gene. The function of this transcription is unknown. An additional layer of transcriptional regulation complexity is due to the fact that the host Dam DNA-(N6-adenine)methyltransferase is required. Dam methylation of three closely spaced upstream GATC sequences is necessary to prevent binding by the host protein, OxyR, which acts as a repressor. Repression is not mediated by inhibition of C binding, but rather through interference with C-mediated recruitment of RNAP to the correct site. Translation of mom is regulated by the phage Com protein. Com is only 62 amino acids long and contains a zinc finger-like structure (coordinated by four cysteine residues) in the amino terminal domain. Com binds mom mRNA 5' to the mom open reading frame, whose translation start signals are contained in a stem-loop translation-inhibition-structure. Com binding to its target site (5' to and adjacent to the translation-inhibition-structure) results in a stable change in RNA secondary structure that exposes the translation start signals.

Bidirectional transcription in the mom promoter region of bacteriophage Mu.

Transcription of the Mu mom operon requires activation by the phage gene product, C, a site-specific DNA binding protein. Previous in vivo and in vitro footprinting studies showed that Escherichia coli RNA polymerase (Esigma70=RNAP) bound the wild-type (wt) mom promoter (Pmom) region in the absence of C; this site, now designated momP2 (-11 to -64), is slightly upstream of, but overlapping with, momP1 (+16 to -49), the functional binding site for mom operon (rightward) transcription. The location/distribution of KMnO4-sensitive sites on the two DNA strands suggested that RNAP bound at momP2 was in an open-complex, but that transcription was in the opposite direction. Here, we used both runoff transcription and reverse transcriptase-primer extension sequencing to provide direct evidence that in the absence of C protein, RNAP carries out leftward transcription from momP2 both in vitro and in vivo. In addition, the 5' ends of these transcripts were mapped to the same upstream initiation site, -58G, relative to the initiation site of C-activated rightward transcription. We also present evidence that leftward transcription from momP2 requires RNAP recognition of an UP-element by the carboxyl-terminal domain of the alpha subunit.

Activation of bacteriophage Mu mom transcription by C protein does not require specific interaction with the carboxyl-terminal region of the alpha or sigma 70 subunit of Escherichia coli RNA polymerase.

Late in its growth cycle, transcription of the phage Mu mom Promoter (Pmom) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the alpha or sigma 70 subunit of Escherichia coli RNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both alpha and sigma 70.

Phage T4 DNA [N6-adenine] methyltransferase: kinetic studies using oligonucleotides containing native or modified recognition sites.

The DNA-[N6-adenine] methyltransferase of T4 phage (T4 Dam MTase) catalyzes methyl group transfer from S-adenosyl-L-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have investigated the effect of eliminating different structural components of the recognition site on the ability of a substrate to be bound and methylated by T4 Dam. For this purpose, steady state binding (by gel shift assays) and kinetic parameters of methylation (using the methyl donor, [3H-CH3]-AdoMet, at 25 degrees C) were studied using various synthetic duplex oligonucleotides containing some defect in the DNA-target site; e.g., the absence of an internucleotide phosphate or a nucleotide(s) within the recognition site, or a single stranded region. The salient results are summarized as follows: (1) Addition of T4 Dam to a complete reaction mixture (with a 20-mer duplex as substrate) resulted in a 'burst' of 3H-methylated product, followed by a constant rate of product formation that reflected establishment of steady-state conditions. This suggests that the rate-limiting step is release of product methylated DNA from the enzyme [and not the transfer of the methyl group]. (2) A number of the defects in duplex structure had only a weak influence on the binding and Km values, but strongly reduced the kcat. At the same time, several poorly bound duplexes retained good substrate characteristics, especially duplexes having uninterrupted GAT-sequences in both strands. Whereas having only one half of the recognition site element intact was sufficient for stable complex formation, the catalytic turnover process had a strict requirement for an uninterrupted GAT-sequence on both strands. (3) There was no correlation between Km and binding capability; the apparent Kd for some duplexes was 5-70 times higher than Km. This indicates that the T4 Dam methylation reaction can not be explained by a simple Michaelian scheme.

Escherichia coli OxyR modulation of bacteriophage Mu mom expression in dam+ cells can be attributed to its ability to bind hemimethylated Pmom promoter DNA.

Transcription of the bacteriophage Mu mom operon is strongly repressed by the host OxyR protein in dam - but not dam + cells. In this work we show that the extent of mom modification is sensitive to the relative levels of the Dam and OxyR proteins and OxyR appears to modulate the level of mom expression even in dam + cells. In vitro studies demonstrated that OxyR is capable of binding hemimethylated P mom , although its affinity is reduced slightly compared with unmethylated DNA. Thus, OxyR modulation of mom expression in dam + cells can be attributed to its ability to bind hemimethylated P mom DNA, the product of DNA replication.

Interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with oligonucleotides containing native or modified (defective) recognition sites.

The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective. The results are summarized as follows. (i) T4 Dam bound with approximately 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, than to a non-specific duplex containing another palindrome, GTAC. (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased ( approximately 2-fold) ability to form complexes with T4 Dam. (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it. This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary. Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues. This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate. Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5'G.C base-pairs be present at both ends of the palindromic, GATC. Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.

Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.

The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.

Interaction of the bacteriophage Mu transcriptional activator protein, C, with its target site in the mom promoter.

The bacteriophage Mu C gene encodes a 16.5 kDa site-specific DNA binding protein that is a transcriptional activator of the four "late" promoters, Pmom, Plys, PI and PP. A symmetrical consensus C recognition sequence, TTAT[N5-6]ATAA, containing an inverted tetrad repeat separated by a spacer of five to six G+C-rich nucleotides, has been proposed. To investigate this, we used oligonucleotide mutagenesis to introduce random substitutions within and flanking the proposed C-target region; each variant site was tested for C recognition by an in vivo functional transactivation assay. We observed that all single mutations, in either tetrad, reduced C activation. Although two out of ten substitutions within the spacer reduced activation, the spacer region does not appear to make specific contact with C. We also used in vitro chemical-protection and -interference to study C contacts with Pmom. The results indicate that C contacts Pmom DNA on only one face of the helix through interactions within two adjacent major grooves; this conclusion was supported by gel shift analyses using synthetic oligonucleotide duplexes containing I.C or other base-pair substitutions. Evidence is also presented that C-Pmom contacts are asymmetrical, and that they extend two nucleotides 3' to the promoter-proximal tetrad. We also show that C binding induces a deformation, possibly a bend, in Pmom DNA.

Escherichia coli OxyR protein represses the unmethylated bacteriophage Mu mom operon without blocking binding of the transcriptional activator C.

Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.

Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase.

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.

Phage T4 DNA [N6-adenine]methyltransferase. Overexpression, purification, and characterization.

The bacteriophage T4 dam gene, encoding the Dam DNA [N6-adenine]methyltransferase (MTase), has been subcloned into the plasmid expression vector, pJW2. In this construct, designated pINT4dam, transcription is from the regulatable phage lambda pR and pL promoters, arranged in tandem. A two-step purification scheme using DEAE-cellulose and phosphocellulose columns in series, followed by hydroxyapatite chromatography, was developed to purify the enzyme to near homogeneity. The yield of purified protein was 2 mg/g of cell paste. The MTase has an s20,w of 3.0 S and a Stokes radius of 23 A and exists in solution as a monomer. The Km for the methyl donor, S-adenosylmethionine, is 0.1 x 10(-6) M, and the Km for substrate nonglucosylated, unmethylated T4 gt- dam DNA is 1.1 x 10(-12) M. The products of DNA methylation, S-adenosyl-L-homocysteine and methylated DNA, are competitive inhibitors of the reaction; Ki values of 2.4 x 10(-6) M and 4.6 x 10(-12) M, respectively, were observed. T4 Dam methylates the palindromic tetranucleotide, GATC, designated the canonical sequence. However, at high MTase:DNA ratios, T4 Dam can methylate some noncanonical sequences belonging to GAY (where Y represents cytosine or thymine).

Studies on the function of conserved sequence motifs in the T4 Dam-[N6-adenine] and EcoRII [C5-cytosine] DNA methyltransferases.

We used site-directed oligodeoxyribonucleotide-mediated mutagenesis and kinetic studies with purified wild-type (wt) and mutant proteins to evaluate the role of the conserved sequence motifs in two prokaryotic DNA MTases. We suggest that: (i) the main role of Pro in the M.EcoRII PC-motif is to restrict the conformational freedom of Cys and orient it in a manner essential for catalysis; (ii) in both M.EcoRII and T4 Dam the FXGXG-motif positions AdoMet with respect to the catalytic site; (iii) the DPPY-motif in T4 Dam (region IV) is important for AdoMet-binding and may be part of the binding site; and (iv) the RXNXKXXFXXPFK-motif in T4 Dam (region III) is part of the DNA binding/recognition domain.

The zinc coordination site of the bacteriophage Mu translational activator protein, Com.

The bacteriophage Mu Com protein is a small "zinc finger-like" protein that binds a specific site in com-mom operon mRNA and activates translation of the mom open-reading-frame. Com contains six cysteine and five histidine residues that have the potential to form several alternative zinc-finger-like motifs. We have used oligonucleotide site-directed mutagenesis to individually alter each of these amino acids (Cys to Ser, and His to Asn or Gln) and tested the various forms of Com for their ability to function in vivo. We observed that mutation of any one of the four N-terminal cysteine residues (Cys-6, 9, 26 or 29) resulted in loss of Com activity. The Com protein requires zinc in order to fold into its functional tertiary structure, as demonstrated by characteristic 1H nuclear magnetic resonance (NMR) chemical shifts. 1H chemical shifts revert to random coil values in the presence of the metal chelator EDTA. The metal-binding specificity and thermal stability of Com also has been investigated using 1H NMR. We report the use of 113Cd NMR, 1H-113Cd heteronuclear spin-echo difference spectroscopy HSED and Zn extended X-ray absorption fine structure spectroscopy EXAFS to determine the zinc/protein stoichiometry as 1:1 and the ligand environment as tetrathiolate. Comparative NMR spectra of Com mutants C6S and C39S suggest position 6 is involved in zinc coordination, while position 39 is not metal-liganded. These studies indicate that the metal coordination, site of Com is a four-cysteine complex, involving residues 6, 9, 26 and 29.