©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Spectroscopic Evidence for a Common Electron Transfer Pathway for Two Tryptophan Tryptophylquinone Enzymes (*)

(Received for publication, August 23, 1994; and in revised form, December 14, 1994)

Steven L. Edwards (§) Victor L. Davidson Young-Lan Hyun Paul T. Wingfield

From the  (1)Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892-2755 (2)Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505 (3)Protein Expression Laboratory, Office of the Director, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Aromatic amine dehydrogenase (AADH) and methylamine dehydrogenase (MADH) are the only two enzymes known to use the cofactor tryptophan tryptophylquinone (TTQ). Each catalyzes oxidative deamination of a distinct class of primary amines. A detailed comparison of their circular dichroic spectra indicates that both proteins share a similar fold with their TTQ cofactors residing in similar environments and that this may be a useful diagnostic probe for TTQ enzymes. Alcaligenes faecalis cells induced to express AADH also express a large amount of the blue copper protein, azurin. Oxidized azurin is rapidly reduced by a catalytic amount of AADH in the presence of the substrate, tyramine. Three A. faecalis cytochromes-c and three other cytochromes-c were tested for electron transfer activity with AADH. Azurin markedly facilitated electron transfer from AADH to each cytochrome. This suggests that AADH and azurin may form an electron transfer complex with a c-type cytochrome, analogous to the crystallographically determined MADH-amicyanin-cytochrome c-551i complex (Chen, L., Durley, R. C. E., Matthews, F. S., and Davidson, V. L.(1994) Science 264, 86-90). The similarities of MADH and AADH plus the demonstration of azurin and multiple cytochromes as functional electron-transfer partners suggest that both TTQ-bearing enzymes share common mechanisms for oxidative deamination and subsequent electron transfer.


INTRODUCTION

A recent advance toward understanding the diversity of redox mechanisms used in nature is the discovery that amino acids can be modified for special catalytic purposes to act as novel cofactors. In a few cases, there is sufficient structural information to unequivocally define the cofactor and its attachment to the surrounding protein. An example is the cofactor, tryptophan tryptophylquinone (TTQ), (^1)which is formed by the covalent attachment of one tryptophan indole ring to a second tryptophan ring with one ring oxidized to an orthoquinone (Fig. 1). This structure was deduced by McIntire et al.(1991) as the cofactor for methylamine dehydrogenase (MADH). How TTQ is formed is still unknown, but it has been shown that the two tryptophans are encoded as normal amino acid residues as determined by the DNA sequence of the MADH gene (Chistoserdov et al., 1990). Recently, we have determined that a related enzyme, aromatic amine dehydrogenase (AADH), also uses the TTQ cofactor and is the second known example of a TTQ enzyme (Govindaraj et al., 1994). AADH is expressed by the soil bacterium, Alcaligenes faecalis, which allows it to break down aromatic amine substrates for use as a source of carbon and energy. These substrates are mainly beta-phenylethylamines in which the phenyl ring is hydroxylated as in dopamine and tyramine, for example (Iwaki et al., 1983).


Figure 1: The structure of TTQ.



Great strides have been made to elucidate the structure of MADH and the TTQ cofactor. Crystal structures have been reported for MADH from Thiobacillus versutus (Vellieux et al., 1986; 1989) and Paracoccus denitrificans (Chen et al., 1991, 1992b). Both structures confirm the alpha(2)beta(2) tetrameric geometry with the cofactor included in the smaller subunit (beta). The larger subunit (alpha) displays the 7-fold, circular repetition of small beta-sheets that was first seen in the structure of influenza neuraminidase (Varghese et al., 1983; Bossart-Whitaker et al., 1993) and more recently in galactose oxidase (Ito et al., 1991) and methanol dehydrogenase (Xia et al., 1992).

Both MADH and AADH catalyze oxidative deamination. In MADH, it has been suggested that the amine moiety of the substrate displaces a quinone oxygen to form an iminoquinone intermediate as part of the mechanism (Backes et al., 1991; Brooks et al., 1993). The participation of TTQ is supported by the crystal structures that show the cofactor near the surface of the beta subunit (near a cleft between the alpha and beta subunits, which would allow an amine substrate to approach the cofactor). Furthermore, hydrazine inhibitors have been used to show that carbon atom C-6 of TTQ is the probable site of reaction (Huizinga et al., 1992). A crystal structure for the binary complex between P. denitrificans MADH and its electron receptor, amicyanin, reveals the copper atom of amicyanin within 10 Å of the TTQ cofactor of MADH (Chen et al., 1992a). Most recently, the structure of a ternary complex of MADH, amicyanin, and cytochrome c-551i has been solved and suggests the geometry for an electron transfer complex involving MADH, amicyanin, and cytochrome c-551i (Chen et al., 1993; 1994).

This wealth of structural information for MADH prompts the questions (i) does AADH have a similar structure to MADH and (ii) does AADH also transfer electrons to a copper protein and/or a cytochrome as part of its catalytic mechanism? To address these questions, we have measured the circular dichroic spectra of AADH and MADH and performed a detailed analysis to determine the similarities of their structures. In addition, we have isolated an azurin and three c-type cytochromes from A. faecalis and tested their ability to function as electron acceptors for the AADH-catalyzed reaction. The ability of AADH and azurin to react with cytochrome c from other sources was also examined. The results of these experiments show that AADH and MADH share many properties and suggest that they may have evolved from a common ancestor.


EXPERIMENTAL PROCEDURES

Materials

Cells of A. faecalis (Institute for Fermentation, Osaka, 14479) were obtained from the Institute for Fermentation (Osaka, Japan) and used for production of the AADH, azurin, and cytochromes. MADH (Davidson, 1990), amicyanin (Husain and Davidson, 1985), and cytochromes c (Husain and Davidson, 1986) were purified from P. denitrificans (ATCC 13543) as previously described.

Electrophoresis

PAGE was performed by standard methods using Novex brand precast gels. Heme staining of gels for c-type cytochromes with dimethoxybenzidine was performed by the method of Francis and Becker(1984).

UV-visible Spectroscopy

Spectra were measured in a 1-cm pathlength cell using a double beam, diode array Hewlett-Packard 8450A UV-visible spectrophotometer. Proteins were reduced with either a few grains of sodium dithionite or with 10 mM tyramine. Protein concentrations were determined using published extinction coefficients for AADH (Govindaraj et al., 1994), MADH (Husain and Davidson, 1987), amicyanin (Husain and Davidson, 1985), azurin (Rosen et al., 1981), and P. denitrificans cytochromes (Husain and Davidson, 1986).

Circular Dichroism Spectroscopy

CD spectra were recorded on a Jasco J-720 spectropolarimeter. Measurements were made using about 1 mg/ml protein in 50 mM sodium phosphate, pH 6.8. In the near ultraviolet region (600-240 nm), a 1-cm pathlength cell and a 1-nm bandwidth were used, and in the far ultraviolet region (260-180 nm), a 0.01-cm pathlength cell and a 1-nm bandwidth were used. Solutions were filtered with Millex-GV 0.22-mm filters (Millipore) and degassed prior to use. Secondary structures were estimated using the methods of Perczel et al.(1990), Sreerama and Woody(1993), and Chang et al.(1978). CD data were expressed as the mean residue ellipticity () in degree cm^2/dmol, with mean residue mass of 109.2 for both AADH and MADH.

Sedimentation Equilibrium

Analytical ultracentrifugation was carried out using a Beckman Optima XL-A analytical ultracentrifuge with an An-60Ti rotor and standard double-sector centerpiece cells. Centrifugation (15 h at 20 °C) of azurin (0.5 mg/ml) in 100 mM potassium phosphate, pH 7.5, was at 23,000 rpm. Data were analyzed using the Beckman-Origin software (v2.0 for Windows). The protein partial specific volume (0.729) for azurin was calculated from the amino acid composition (Cohn and Edsall, 1943) using the sequence published by Ambler(1971). The solvent density was calculated as described by Laue et al.(1992).

Protein Sequencing

NH(2)-terminal amino acid sequencing was carried out by automated Edman degradation using the Applied Biosystems model 477A protein sequencer. Identification and quantitation of the phenylthiohydantoin derivatives were performed on line using an Applied Biosystems model 120A PTH-derivative analyzer.

Isolation of Azurin

The isolation of azurin from A. faecalis has been described before (Ambler, 1971). Initially, it was not clear that the blue protein observed was azurin. Consequently a different isolation procedure was developed, which yielded high purity protein. Analysis of the purified protein allowed it to be identified as azurin.

Isolation of a High Molecular Weight Cytochrome c

The green, AADH-containing fraction from the initial DEAE anion exchange column previously described (Govindaraj et al., 1994) was applied to a Pharmacia Sephacryl S-200 column (2.5 times 90 cm). A pink band separated from the green band (which is predominantly AADH) and eluted shortly after it. Pink fractions were collected, and their absorption spectra showed a sharp peak at 408 nm and a broad band around 550 nm. On the addition of sodium dithionite, the major peak shifted to 418 nm, and the secondary band changed to show distinct peaks at 523 and 553 nm; these spectral changes indicated that the pink protein probably contained a c-type cytochrome. The cytochrome was dialyzed against 10 mM Tris, pH 7.5, and applied to a DEAE column equilibrated with the same buffer. The cytochrome bound to the top of the column and began to spread as the Tris concentration was gradually increased to 50 mM. The cytochrome was eluted with 10 mM NaCl added to the column buffer. The cytochrome containing fraction was dialyzed against 50 mM sodium acetate, pH 4.5, and applied to a column of CM-Sepharose equilibrated with the same buffer. The protein bound weakly and eluted with the column buffer. SDS-PAGE of the cytochrome is shown in Fig. 2, and its absorption spectrum is shown in Fig. 3.


Figure 2: SDS-PAGE of the large cytochrome from A. faecalis (lane 2), aromatic amine dehydrogenase (lane 3), and azurin (lane 4) analyzed on a 4-20% gradient gel. In lane2, the large cytochrome band at 38 kDa stained positively for heme; a faint band just below it did not. The molecular weight standards (Bio-Rad) in lane1 are phosphorylase B, 97,400; bovine serum albumin, 66,200; ovalbumin, 42,699; bovine carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,500; and hen egg white lysozyme, 14,400.




Figure 3: Comparison of oxidized (solid line) and reduced (dotted line) forms of AADH (reduced by the addition of tyramine) (A), azurin (reduced with sodium dithionite) (B), and large cytochrome (reduced with sodium dithionite) (C). All of these proteins were isolated from the same growth of A. faecalis cells (Institute for Fermentation, Osaka, 14479) induced to express AADH with beta-phenylethylamine. Spectra of the reduced forms of azurin and the cytochrome are identical to ones resulting from AADH-catalyzed oxidation of tyramine.



Identification of Other Cytochromes c

At least two other cytochromes were observed during the purification of AADH. Two pink fractions eluted from the DEAE column, well separated from the other proteins of interest. Analysis of each fraction by SDS-PAGE indicated that the cytochromes were only a minor component. Heme staining of the gels indicated that the first pink fraction to elute from the column (fraction I) contained a cytochrome of approximately 15 kDa. The second pink fraction (fraction II) contained a cytochrome of approximately 25 kDa with a faint heme staining band in the region of 35-40 kDa. The amount of each cytochrome that was recovered from the DEAE column was very small relative to the amounts of AADH and azurin. Because of the low yields of each cytochrome, it was not possible to further purify these fractions.


RESULTS AND DISCUSSION

The electron transfer pathway for oxidative deamination involving the TTQ enzyme methylamine dehydrogenase is well understood biochemically, and the proteins that probably link this pathway to cytochrome oxidase have been elucidated with crystal structures (Chen et al., 1994). In contrast, the electron transfer partners for aromatic amine dehydrogenase were previously unknown, and possible structural similarities between AADH and MADH were poorly understood. We describe here proteins from A. faecalis that appear to parallel the electron transfer pathway from MADH to c-type cytochromes in P. denitrificans and present circular dichroic spectra that suggest strongly that the overall fold of AADH is like that of MADH and in particular that the environments of both TTQ cofactors are similar.

Circular Dichroic Spectroscopy of AADH and MADH

The circular dichroic spectra of AADH and MADH are quite similar (Fig. 4). The far UV-CD spectra of both proteins exhibit a broad trough of negative ellipticity with minima near 215 and 198 nm and a positive band at 187 nm (Fig. 4A). From the spectra, the secondary structure of the proteins was estimated by three methods (Table 1). The combined beta-sheet and beta-turn contents of both proteins are around 60-65%, and the average alpha-helix content is 6-8%. These estimates can be compared with the values derived from the crystal structure of MADH, namely 52% beta-sheet and 5% alpha-helix. (^2)Although the determination of secondary structure from CD spectra is of course not as rigorous as that by direct determination, the similarity of the two dehydrogenase spectra and secondary structure elements obtained from their decomposition does suggest related overall structures. We note also that the CD spectrum for viral neuraminidase (Baiocchi et al., 1993) is also similar in shape to the dehydrogenase spectra, consistent with the proposal that AADH and neuraminidase have a common fold (specifically, the circular repetition of multiple four-stranded beta-sheets).


Figure 4: Circular dichroic spectra of AADH and azurin from A. faecalis compared with MADH and amicyanin from P. denitrificans. A, far UV spectra of AADH and MADH; B, far UV spectra of azurin and amicyanin. C, near UV-visible spectra for oxidized and reduced MADH; D, near UV-visible spectra for oxidized and reduced AADH. The insets of C and D show the oxidized minus reduced difference spectra for MADH and AADH, respectively. The ellipticity, (mean residue weight), is in units of degree cm^2/dmol.





We also compared the CD spectra of both proteins in the visible and near UV regions to assess the similarity of aromatic groups, including the TTQ cofactor (Fig. 4, C and D). The spectra of the oxidized proteins are very similar, and strong positive ellipticities were observed around 450, 290, and 262 nm. The band at 450 nm corresponds to the absorption peak (Fig. 3A) of the oxidized quinone moiety of the TTQ chromophore. Reduction of the dehydrogenases with dithionite in both cases caused loss of all three major bands with the appearance of positive bands at 333 and 290 nm (MADH) and 335 and 294 nm (AADH). These changes mirrored those in the UV-visible absorbance upon reduction (Fig. 3). A negative CD band at 272 nm (AADH) and 277 nm (MADH) was also observed. The signals at 272-277 and 290 nm are presumably derived from asymmetrically oriented tyrosine and tryptophan residues, respectively (Strickland, 1974). The oxidized minus reduced CD difference spectrum was nearly identical for both proteins (Fig. 4, C and D, inserts).

The near UV-visible CD spectral region of the dehydrogenases is clearly dominated in the oxidized proteins by the TTQ chromophore. The band positions and intensities are comparable enough to suggest that the orientation of TTQ is similar in both proteins. Denaturation of the oxidized proteins with 4 M guanidine HCl resulted in loss of all CD bands, except a weak negative one around 277-275 nm (data not shown). This band appears to correspond to the band seen in the reduced protein, although the intensity of the former is only about 15% of that in the native reduced proteins. From the above results, MADH and AADH have similar far UV-CD spectra consistent with related secondary structures. In the aromatic and visible regions, they also have similar CD spectra in the oxidized, reduced, and denatured states, suggesting homologous tertiary structures.

Identification of Azurin as Electron Acceptor for AADH

Large amounts of a blue protein were also expressed with AADH when A. faecalis cells were grown on beta-phenylethylamine as a carbon source. The identity of the blue protein was suggested by its visible spectrum. A broad absorption peak centered at 625 nm and a minor one at 417 (at low pH) are characteristic of azurin. To confirm this, amino-terminal peptide sequencing was performed on the first 40 residues. 37 residues identically matched the reported sequence for azurin from A. faecalis (Ambler, 1971); the remaining residues did not give a clear signal. Two of these residues were at the 2nd and 25th positions, which correspond to cysteines in the published sequence (cysteines were not detectable in our sequence analysis), and the third was at position 37, which corresponds to His. The molecular weight, as determined by sedimentation equilibrium (13.2 kDa), is within experimental error of the value for azurin calculated from the weights of amino acids reported in its sequence (13,393 Da). We note that the molecular mass estimated from the SDS-PAGE analysis (about 17 kDa) was anomalously high (Fig. 2). As azurin is not known to contain any posttranslational modifications, this anomalous behavior may be due to the content and distribution of charges on the protein. These data suggest strongly that the blue protein is an azurin, probably similar if not identical to the azurin previously reported for A. faecalis strain, NCIB 8156 (Ambler, 1971). Its circular dichroic spectrum was consistent with the secondary structure observed in the crystal structure of azurin from Alcaligenes denitrificans, which has recently been reclassified as A. faecalis NCTC 8582 (Baker, 1988). It is clearly distinct from the circular dichroic spectrum measured from amicyanin (Fig. 4B). The main structural difference between the two is that azurin has two alpha helices in addition to the eight-strand beta-sheet core common to all type I copper proteins (Adman, 1991), whereas amicyanin has no helices (Durley et al., 1993). It is likely that the azurin described in this report will be structurally similar if not identical to the crystal structure already reported (Baker, 1988).

The functional role of azurin was investigated by testing its ability to receive electrons from AADH. Incubation of oxidized azurin with either oxidized AADH or tyramine alone caused no change in the redox state of azurin. However, when 5.5 µM azurin was incubated with 0.033 µM AADH in 0.1 M potassium phosphate buffer, pH 7.5, addition of 50 µM tyramine caused complete reduction of azurin as indicated by the complete loss of absorbance at 625 nm. Under these non-saturating conditions, the initial rate of azurin reduction was 960 min.

A. faecalis has been reported to express both an azurin and a pseudoazurin, which participate in various electron transfer pathways. For example, azurin has been identified as the electron acceptor for the molybdenum-containing enzyme, arsenite oxidase (Anderson et al., 1992), while pseudoazurin acts as an electron donor for nitrite reductase (Kakutani et al., 1981). Curiously, it was observed that pseudoazurin in an aerobic solution containing ascorbate or cysteine but lacking nitrite caused inactivation of nitrite reductase. A suicide mechanism involving the production of peroxide has been proposed (Kakutani et al., 1981). It should be noted that no copper protein other than azurin was observed during the purification of proteins from the beta-phenylethylamine-grown cells of A. faecalis.

Properties of a Large c-type Cytochrome

A large c-type cytochrome (M(r) = 38,000) was observed, which tended to co-purify with AADH. Our efforts to isolate it from AADH resulted in large losses due to irreversible binding of the cytochrome to various chromatographic resins. AADH and azurin persisted as contaminants. An additional protein with M(r) = 25,000 could not be separated from the cytochrome (Fig. 2).

The protein was analyzed by SDS-PAGE; the gel was lightly stained with Coomassie Blue and subsequently analyzed for heme with dimethoxybenzidine (Francis and Becker, 1984). The band at 38 kDa stained positively (green), indicating the presence of covalently bound heme. Control experiments with the same stain were positive for horse heart cytochrome c and negative for cytochrome c peroxidase, which has a noncovalently bound heme (data not shown).

The oxidized form of the cytochrome has a prominent absorption peak at 408 nm and a broad band centered around 550 nm (Fig. 3). It could be reduced by the addition of a few grains of solid sodium dithionite, which shifted the soret peak to 418 nm and induced the appearance of two peaks at 523 and 553 nm. These spectral properties and the covalent attachment of a heme are characteristic of c-type cytochromes. A cytochrome with similar spectral properties was observed in the study of arsenite oxidase from A. faecalis (Anderson et al., 1992). Whether this is the same protein cannot be determined at present since the cytochrome described in that study was not extensively purified. (^3)Amino terminal peptide sequencing of the first 20 residues showed no homology to any known cytochrome. (^4)

A sample of the cytochrome was used to test its ability to receive electrons from AADH. When the cytochrome was incubated with tyramine and AADH alone, no change in the spectrum occurred. When azurin was included in the same reaction mixture, but prior to adding the cytochrome, the spectrum quickly changed to be identical to that of the reduced cytochrome (Fig. 3). However, when azurin was added after the cytochrome had been incubated with AADH, the spectrum of the cytochrome slowly changed, showing the appearance of the 523- and 553-nm peaks, but the soret peak shifted only slightly from 408 to 410 nm. In both cases, when the reduction of the cytochrome appeared complete, a minimal amount of the oxidizing agent potassium ferricyanide was added, which caused full reoxidation of the cytochrome as judged by its spectrum. Subsequently, full reduction could be achieved by adding a few grains of dithionite.

Mediation of Electron Transfer by Azurin from AADH to c-type Cytochromes

As discussed earlier, it was possible to obtain small amounts of two other crude cytochrome-containing fractions from the extract of A. faecalis cells, which had been grown on beta-phenylethylamine. The ability of the cytochromes in these fractions to accept electrons from AADH and azurin was examined (Table 2). When cytochrome fraction I was incubated with AADH and tyramine, no reduction of the cytochrome was observed. Relatively slow reduction of the cytochrome was only observed on addition of azurin. The cytochrome fraction II exhibited autoreduction in the presence of oxidized AADH with no substrate present. Addition of tyramine caused ony a very slight increase in the rate of cytochrome reduction above the autoreduction rate. However, addition of azurin led to a marked increase in the rate of cytochrome reduction. The significance of the observed autoreduction of this cytochrome fraction is unclear. It will be of interest to examine this phenomenon further when the cytochrome has been completely purified and better characterized.



Three well characterized cytochromes from other sources, horse heart cytochrome c and cytochromes c-550 and c-551i from P. denitrificans, were also tested for electron transfer ability (Table 2). With two of these cytochromes, very little reaction occurred when each was incubated with AADH and tyramine. On addition of azurin, the rates increased approximately 50-fold for horse heart cytochrome c and 100-fold for cytochrome c-550. Cytochrome c-551i showed significant electron transfer ability with AADH in the absence of azurin but also exhibited a 4-fold stimulation in rate when azurin was present. Thus, with each of the c-type cytochromes that were examined, azurin was demonstrated to mediate electron transfer from AADH, and with most of the cytochromes, azurin was absolutely required to observe any significant tyramine-dependent cytochrome reduction by AADH.

Electron transfer interactions between blue copper proteins and cytochromes have long been recognized as some of the many complex redox pathways manifested by denitrifying bacteria. In P. denitrificans, amicyanin may transfer an electron to cytochrome c-550 or c-551i after reduction by MADH (Husain and Davidson, 1986). These cytochromes presumably transfer electrons to cytochrome oxidase. A similar system appears to operate in Alcaligenes. As shown here, azurin is capable of mediating electron transfer from AADH to three endogenous cytochromes. Cytochromes from P. denitrificans were also able to accept electrons from azurin in the AADH-catalyzed reduction of tyramine. We note that a variety of non-physiological combinations of azurins with cytochromes c have already been studied as model systems for investigating structural aspects of electron transfer processes (Wherland and Pecht, 1978). The new information presented here will provide a basis for future studies of interactions between this azurin and its physiological as well as non-physiological redox partners.

Concluding Remarks

The TTQ-bearing enzymes AADH and MADH are members of a new class of enzymes that catalyze oxidative deamination of primary amine compounds to provide energy and assimilate carbon for their host cells. MADH has been extensively studied biochemically (reviewed in Davidson(1993)), and detailed structural information has come from x-ray crystallography (Chen et al., 1992a, 1992b). Taking advantage of this, we have made circular dichroic measurements on AADH and compared them with MADH to deduce structural information about AADH. The spectra show significant similarites, indicating that they have similar secondary structures and probably the same general and rather distinct fold. Spectra measured in the near UV-visible region of reduced and oxidized forms of the proteins indicate that the TTQ environments are also similar. This spectroscopy provides a foundation for examining TTQ dehydrogenases and may assist in the discovery of new ones.

In addition, the current experiments show that AADH uses the blue copper protein, azurin, to mediate electron transfer to cytochromes homologous to the role of amicyanin in P. denitrificans. These structural and functional similarities suggest that AADH and MADH may have evolved from a common ancestor to yield enzymes with distinct substrate specificities. Information for understanding this specificity is suggested by a new high resolution structure of MADH.^2 It shows that the methylamine binding pocket is small, which helps to explain why MADH does not accept large substrates like tyramine. AADH probably has a different binding pocket geometry designed to accept larger substrates, and it will be interesting to see how this binding pocket selects against methylamine as a substrate. A final speculation is prompted by the recently determined crystal structure of a ternary complex between MADH, amicyanin, and cytochrome c-551i (Chen et al., 1994). Assuming that AADH would form a similar complex with azurin and a cytochrome, how would the known difference in structure between azurin and amicyanin affect it? The most significant difference is that azurin has two helices near the copper binding site missing in amicyanin. The position of these helices would not appear to affect the interaction with AADH but would more likely affect the interaction with a cytochrome. Hopefully, more direct information will come from crystal structures of AADH and its complex with azurin and from solution studies of the electron transfer reaction between AADH and azurin, which we are currently pursuing.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM41574 (to V. L. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Section of Molecular and Cellular Biology, Briggs Hall, Rm. 149, University of California, Davis, CA 95616. Tel.: 916-752-6417, Fax: 916-752-3085.

(^1)
The abbreviations used are: AADH, aromatic amine dehydrogenase; MADH, methylamine dehydrogenase; TTQ, tryptophan tryptophylquinone; PAGE, polyacrylamide gel electrophoresis.

(^2)
L. Chen, personal communication.

(^3)
R. Hille, personal communication.

(^4)
P. T. Wingfield, unpublished results.


ACKNOWLEDGEMENTS

We express our gratitude for support from C. Craig Hyde of the Laboratory of Structural Biology Research, NIAMS, in whose laboratory much of this work was performed. We thank Shanthi Govindaraj at the University of California, Irvine (Dept. of Biochemistry and Molecular Biology) for growing some of the A. faecalis cells used in this experiment. We also thank Prof. Elinor Adman at the University of Washington (Seattle) for insightful comments regarding blue copper proteins. We are grateful to Dr. Longyin Chen and F. Scott Matthews and colleagues at the Washington University School of Medicine (St. Louis, MO) for sharing the results of refinement on MADH prior to publication.


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