Isolation, Characterization, and Amino Acid Sequences of Auracyanins, Blue Copper Proteins from the Green Photosynthetic Bacterium Chloroflexus aurantiacus”

Three small blue copper proteins designated auracyanin A, auracyanin B-l, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7868-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electropho-resis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the elec- tron band Isolated membranes "C to the auracyanin. irradiated The sample was in and A light-minus- dark difference spectrum was obtained by subtracting the dark sample spectrum from the sample spectrum. Similar experiments were carried out on salt-washed (1 M NaCI, 100 mM MgC12) membranes (Am = 41.7). Additionally, a chemically induced signal was obtained by adding 1 millimolar potassium ferricyanide. The continuous illumination optical study was carried out on isolated membrane samples (Asas = 13.7) which had been treated with 200 mM NaCl and 20 mM M&l2 to remove the majority of the auracyanin. The membranes were placed in 20 mM Tris, pH 8.0, with 200 p~ ascorbate and 1 mM MgC12. Reduced auracyanin was added to a final concentration of 8.36 p ~ . The experiment was carried out using a Shimadzu spectrophotometer equipped with a light for illumination of the sample cuvette. The membranes were illuminated through a light for 5 s by actinic white light from 100-watt tungsten six data points were collected for the determination of second order rate constants. Six ionic strengths were used to measure the dependence of second order rate constants on ionic strength for reaction with fully reduced FMN as described previously (Tollin et al., 1986). Pseudo-first order conditions were maintained for the reaction of reduced auracyanin (100 p ~ ) with oxidized cytochrome c-554 (8 p ~ ) . There was only sufficient protein available to obtain kinetic data at two concentra- tions to ensure that the auracyanin:c-554 reaction was second order, and only one ionic strength (96 mM) was studied for this reaction.

Three small blue copper proteins designated auracyanin A, auracyanin B -l , and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.
Single copper-containing proteins collectively known as 11 To whom correspondence should be addressed. Tel.: 602-965-1439;Fax: 602-965-2747. blue copper proteins or cupredoxins are found in many different organisms. These proteins are characterized by a molecular mass of 10-22 kDa and an intense absorption band near 600 nm in the oxidized form (Rydin, 1984;Adman 1985). They contain a type 1 copper(I1) ion characterized by an intense blue color and an ESR' signal with g values similar to simple copper (11) chelates with half their value for the hyperfine splitting constants (Boas, 1984).
The blue copper proteins have been widely studied and recent reviews have compared the structures  and resonance Raman spectra (Han et al., 1991) of several different proteins. They have been divided into seven distinct classes (Rydin, 1988) with the auracyanin blue copper proteins described in this work appearing to form an eighth class. Most of the proteins are thought to function in electrontransfer processes (Rydin, 1984). The plastocyanins function in most oxygen-evolving photosynthetic systems to transfer electrons from the cytochrome b,$ complex to the reaction center of Photosystem I (Haehnel, 1986). In the anoxygenic photosynthetic bacteria, C-type cytochromes have heretofore always been found to function in the analogous position in the electron transport chain, transferring electrons from the cytochrome bcl complex to the single bacterial reaction center (Dutton, 1986). Certain cyanobacteria and green algae contain either a C-type cytochrome or a plastocyanin at this point in the electron transfer chain (Sandmann et al., 1983). The control of which protein is expressed depends on copper availability. (Wood, 1978;Sandmann and Boger, 1980;Merchant and Bogorad, 1986a, 198613).
Chloroflexus aurantiacus is a green gliding thermophilic photosynthetic bacterium (Pierson and Castenholz, 1974), which is only distantly related to other photosynthetic organisms, according to 16 S rRNA analysis (Woese, 1987). It appears to branch away from the eubacteria very early, and has therefore been suggested as possibly representative of the earliest photosynthetic cells. However, it has recently been suggested that C. aurantiucus may have acquired its photosynthetic capability by lateral gene transfer (Beanland, 1990;Blankenship, 1992). Previous studies have noted an apparent lack of soluble C-type cytochromes, and the identity of the putative electron carrier that completes the cyclic electron transfer pathway has not been identified. (Bartsch, 1978;Bruce et al., 1982;Wynn et al., 1987). C. aurantiacus contains The abbreviations used are: ESR, electron spin resonance; BNPS, 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography. a tetraheme membrane-bound cytochrome that functions as the immediate electron donor to the reaction center (Bruce et al., 1982;Freeman and Blankenship, 1990;Dracheva et al., 1991). This cytochrome is similar in both function and sequence to the tetraheme cytochromes found in many purple photosynthetic bacteria (Deisenhofer and Michel, 1989;Dracheva et al., 1991).
We have characterized three distinct copper-containing proteins (designated auracyanin A, auracyanin B-1, and auracyanin B-2) isolated from C. aurantiacus. Isolation and some properties of the A form were previously described by Trost et al. (1988). The sequence of the A form is presented in van Beeumen et aL2 EXPERIMENTAL PROCEDURES Materials-All chemicals used were reagent grade and obtained from commercial sources unless specified otherwise. Ultrapure grade SDS was obtained from Boehringer Manheim. Acrylamide and bis acrylamide were from Bio-Rad.
Cell Growth and Protein Purification-A 16-liter glass-walled fermenter was used to grow C. aurantiacus strain J10-fl under high light conditions at 55 "C in the modified medium D of Pierson and Castenholz (1974). Approximately 200 g of wet-packed cell paste was brought to a volume of 300 ml with 20 mM Tris-C1, pH 8.0, 0.02% NaN3. The cell suspension was cooled to 0 "C with an ice bath and sonicated three times for 3 min each at a power setting of 8 on a Branson Model 350 sonifier cell disrupter. During the second sonication, the protease inhibitor phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM from a 100 mM stock solution in isopropanol. Unbroken cells and cell debris were removed by centrifugation at 12,100 X g for 15 min. Membranes were pelleted by centrifugation of the resulting supernatant liquid at 200,000 X g for 120 min in a Beckman Ti-50.2 rotor or for 180 min in a Beckman Ti-40.5 rotor. The pelleted membranes were resuspended with 20 mM Tris, 0.02% NaN3, pH 8.0, and diluted to an optical density of 15 at 865 nm.
In most experiments, auracyanin was obtained by salt treatment of the isolated membranes. Diluted membranes were stirred overnight at 4 "C in 1 M NaCl and 100 mM MgC12. In later preparations, a 0.2% final concentration of the detergent lauryldimethylamine N-oxide was added to the membranes in conjunction with the salt treatment to increase the yield of auracyanins. These treatments were followed by ultracentrifugation at 200,000 X g. The supernatant liquid from the ultracentrifugation was fractionated by adding solid (NH4),S04.
Additions of 10% w/v were made with stirring at 0 "C followed by a 15-min incubation period. Precipitated material was removed after each addition by centrifugation at 12,100 X g for 10 min. Crude auracyanin precipitated between approximately 30 and 40% w/v (NH&SO4, as assayed by oxidized (plus ferricyanide) minus reduced (plus ascorbate) visible absorbance difference spectroscopy. The pellet containing the auracyanin was resolubilized in 20 mM Tris, pH 8.0, and dialyzed against the same buffer to remove (NH,),SO,. After concentration using an Amicon PM-10 or YM-10 membrane filter, the sample was chromatographed on a 2.5 X 35-cm Sephadex G-100 gel filtration column. The auracyanin-containing fractions were pooled, reconcentrated, and chromatographed on DEAE-Sephacel in 20 mM Tris, pH 9.0 using a 0-40 mM NaCl step. Additional purification was carried out on a 24 X 1.8-cm wide hydroxylapatite column using 1 mM phosphate, pH 7.0, as the elution buffer. The auracyanin samples did not stick to the column.
Biochemical Characterization and Physical Properties-Molecular weights were determined by two different methods. Discontinuous SDS-PAGE was done using samples treated with 2% SDS and heated at 100 "C for 5 min either with or without 10 mM dithiothreitol (DTT) on 12.5% T gels or with samples heated at 65 "C for 30 min and run on a 15% T gel. The apparent molecular weights of the auracyanin forms were estimated by comparison with molecular weight standards from Sigma. They were also determined using a calibrated Sephadex G-100 gel filtration column. The standards used were a-chymotrypsinogen A type I1 from bovine pancreas (25.7 kDa), bovine serum albumin (66.0 kDa), horse heart cytochrome c (12.4 kDa), and carbonic anhydrase from bovine erythrocytes (29.0 kDa). The auracyanin samples were run with and without 10 mM DTT in 20 mM Tris, pH * J. J. van Beeumen, W. Hu, G. van de Werken, J. D. McManus, and R. E. Blankenship, manuscript in preparation. 8.0, and also in this same buffer without DTT but in the presence of 200 mM NaC1. The isoelectric point of all three proteins was determined using a narrow range (pH 2-7) polyacrylamide isoelectric focussing gel.
Copper content was determined by the method of standard additions using a Perkin-Elmer Model 3300 atomic absorption spectrophotometer equipped with a Model HGA 700 graphite furnace and an AS-70 autosampler. Absorption was monitored at 324.8 nm by the multielement hollow cathode lamp using deuterium arc background correction. The auracyanin samples and dilution buffer (10 mM Tris, pH 8.0) used for copper analysis were passed through a 2 X 23-cm column of Chelex 100 resin (Bio-Rad) to remove free copper and the absorption spectrum uersus 10 mM Tris, pH 8.0, was recorded using a Cary 219 spectrophotometer. Protein assays were carried out in triplicate using the bicinchoninic acid method (Pierce Chemical Co.) with bovine serum albumin as the standard.
Potentiometric titrations of auracyanins B-1 and B-2 were carried out in a homemade four-necked glass titration vessel on samples with AeW = 0.4 in 20 mM Tris, 0.02% NaN3, pH 8.0. The absorption spectrum was recorded as the sample was pumped through a 1-cm path length flow cell. The potential was referenced to a Radiometer K401 calomel electrode with a Radiometer PlOl platinum working electrode, measured using an Orion Model 501 digital ionanalyzer. Sodium ascorbate and potassium ferricyanide were used as titrants.
Analysis for sugar was carried out by two methods. Direct staining of the proteins and membrane samples in a 15% T SDS-PAGE gel was done using a thymol/sulfuric acid method (Gender, 1984). The phenol/sulfuric acid method (Dubois et al., 1956) was used to assay for neutral sugar content of all three isolated auracyanin forms. a-Acid glycoprotein, cytochrome c, and ovalbumin were used as controls.
Amino Acid Composition-A Hewlett-Packard Amino Quant amino acid analyzer was used for the amino acid composition determination of purified protein and peptides. A 24-h vapor-phase hydrolysis of the samples was done using 6 N HCl with 1% phenol. Amino acids in the hydroylsate were derivatized with o-phthaldialdehyde and fluorenylmethyl chloroformate and quantified using fluorescent peak areas.
Sequence Determination-The auracyanin B-1 and B-2 samples were further purified and denatured using a 4.6-mm by 5-cm VYDAC C4 HPLC column. A 5-60% gradient of 70% formic acid, and 70% formic acid containing 30% isopropyl alcohol was run over a 18-min time period. Some of the samples were reduced and carboxymethylated before undergoing protease or chemical treatment (Crestfield et al., 1963).
A Porton Instruments Model PI-2090 gas phase protein sequenator was used for the automated Edman degradation sequencing of the protein samples. The HPLC-separated peptides were sequenced from fiberglass peptide disks, while electrophoretically separated peptides were electroblotted onto immobilon. The short C-terminal cyanogen bromide fragment was covalently attached to a Sequalon AA membrane disk (Millipore) for sequencing. A 10-pl aliquot of o-phthaldialdehyde was added to some samples to reduce the amount of background and lag after sequencing the peptide to a known proline. The o-phthaldialdehyde solution was 1% in the Porton S1 solvent (ethylacetate with DTT). This differed slightly from the method of Brauer et al. (1984).
Generation and Separation of Peptides for Sequencing-The N termini of both B forms of auracyanin were determined by direct application of the intact protein onto Porton fiberglass protein disks. one of three methods. Two different SDS-PAGE gel systems (Schag- The peptides from the protease-treated proteins were separated by ger and Von Jagow, 1987) were used. One gel was a straight 16% T, 3% C with a 10% T, 3% C spacer gel. The other gel was a 10-25% T, 3-0.75% C gradient gel. The proteins were blotted onto immobilon for 45 min at 90 V using a Hoefer Transfor Unit and a buffer consisting of 20% methanol, 25 mM Tris, and 192 mM glycine, pH 8.2 (Matsudaira, 1989). Other samples were run over a 4.6-mm by 15cm VYDAC CIS column. The HPLC solvents and gradients used for separation are outlined below.
A 100-fold molar excess of cyanogen bromide over methionine residues was added to the B-1 and B-2 auracyanin samples which had been dissolved in 70% formic acid. The reaction was carried out for 12 or 24 h at room temperature (25 "C) in the dark. The resulting peptides were separated by HPLC using a flow rate of 1 ml/min with a 0-40% gradient over 45 min immediately followed by a 40-80% gradient over 15 min. The solvents used were 0.1% trifluoroacetic acid and 80% acetonitrile, 0.1% trifluoroacetic acid. The peptides were also separated by SDS-PAGE using 16% gels.
The auracyanin samples for trypsin and chymotrypsin cleavage were first denatured by the addition of 8 M urea, 5 mM DTT in 0.4 M NH4HCOa, pH 8.0 (Matsudaira, 1989). The samples were heated at 50 "C for 15 min and after cooling back down to room temperature, iodoacetamide was added to a final concentration of 8.3 mM. Protease was added to a 1/25 enzyme-to-protein ratio (w/w), and the samples were allowed to incubate at 37 "C for 24 h. The usual size for the auracyanin sample was 100 pg. The resulting peptides were separated by SDS-PAGE using 16% gels.
A BNPS-skatole stock solution that was 6.9 mg/ml in 50% acetic acid was prepared. To approximately 1-2 nmol of protein, 40,50,60, 70, or 90 pl of the BNPS-skatole stock solution was added. The samples were incubated in the dark at room temperature for times varying from 24 to 48 h.
The hydroxylamine treatment buffer was 2 M hydroxylamine in 6 M guanidine HCI, pH 9.5. The correct pH was maintained by adding either LiOH or NaOH. The sample was reacted for 3 h at 45 'C. The resulting peptides were separated by HPLC using a flow rate of 1 ml/ min with a 0-40% gradient over 45 min immediately followed by a 40-80% gradient over 15 min. The solvents used were 0.06% trifluoroacetic acid and 80% acetonitrile, 0.052% trifluoroacetic acid. The samples were also run on SDS-PAGE gradient gels after passing through a Sephadex G-25 desalting column.
The hydroxylamine cleaved at the expected site, but the resulting peptides could not be separated by either SDS-PAGE or HPLC. The longest incubation times used for each treatment resulted in the highest amount of cleavage. Reduction and carboxymethylation did not seem to affect the protease or chemical cleavages.
For clostripain protease treatment, the protein samples were dissolved in 50 pl of 6 M urea, 50 mM Tris, pH 8.0, 5 mM DTT with 0.1% SDS. The samples were heated at 60 'C for 1 h. Clostripain was added (2 pg), and the samples were incubated at 37 "C for 24 h. The resulting peptides were separated by the SDS-PAGE gradient gels and blotted onto Immobilon for sequencing. Spectroscopy-A Shimadzu UV-160 or a Cary 219 recording UVvis spectrophotometer was used to take absorption spectra. The Vis/ NIR circular dichroism (CD) spectrum was recorded on an instrument described elsewhere (Brune et al., 1987;. Raman spectra were obtained on a computer-interfaced Jarrell-Ash spectrophotometer equipped with a Spectra Physics 2025-11 (Kr) laser, an RCA C31034 photomultiplier tube, and an ORTEC Model 9302 amplifier/discriminator. The Raman spectra were collected in an -150" backscattering geometry with samples frozen on the gold-plated cold head of a closed-cycle helium refrigerator (Air Products Displex) maintained at 15 K. For equilibration in D20, a sample of 1 mM auracyanin A was diluted 20-fold in 20 mM Tris in D20 (pH 8.0), then reconcentrated in a Centricon 10 (Amicon). This procedure was repeated two times, including an overnight incubation at 5 "C prior to the final concentration step.
ESR spectra were taken on a Bruker ER2OOD spectrophotometer in quartz sample tubes at both X and Q bands. The temperature of the samples was maintained at either 77 or 4 K. Computer simulations of the auracyanin A and B ESR spectra were done using a program adapted from Daul et al. (1981). Electron spin resonance spectra of membranes were taken at 77 K on a Bruker ER200D spectrophotometer in quartz sample tubes at X band frequency. Isolated membranes (Asas = 41.0) of C. aurantiacus were incubated with 700 p~ ascorbate at 0 "C for 1 h to reduce the auracyanin. One sample was irradiated with actinic white light from a 100-watt tungsten lamp for 2 min before freezing in liquid nitrogen. The second sample was kept in the dark and frozen. A light-minusdark difference spectrum was obtained by subtracting the dark sample spectrum from the light sample spectrum. Similar experiments were carried out on salt-washed (1 M NaCI, 100 mM MgC12) membranes ( A m = 41.7). Additionally, a chemically induced signal was obtained by adding 1 millimolar potassium ferricyanide. The continuous illumination optical study was carried out on isolated membrane samples (Asas = 13.7) which had been treated with 200 mM NaCl and 20 mM M&l2 to remove the majority of the auracyanin. The membranes were placed in 20 mM Tris, pH 8.0, with 200 p~ ascorbate and 1 mM MgC12. Reduced auracyanin was added to a final concentration of 8.36 p~. The experiment was carried out using a Shimadzu UV-160 spectrophotometer equipped with a light pipe for illumination of the sample cuvette. The membranes were illuminated through a light pipe for 5 s by actinic white light from a 100-watt tungsten lamp. The light passed through a Corning 496 cut-off filter before reaching the sample and the absorbance was monitored after the signal passed through a Hoya 054 cut-off filter. The absorbance of illuminated membranes with and without added auracyanin was monitored at 5-nm intervals from 540 to 605 nm.
Kinetics-The laser flash photolysis apparatus and methods of kinetic data acquisition and analysis were as previously described (Tollin et al., 1986). The pseudo-first order kinetics of oxidation of reduced flavin and reduction of auracyanin were followed at 600 nm over several half-lives under anaerobic conditions. The concentration of auracyanin was varied from 8 to 60 p~, and four to six data points were collected for the determination of second order rate constants. Six ionic strengths were used to measure the dependence of second order rate constants on ionic strength for reaction with fully reduced FMN as described previously (Tollin et al., 1986). Pseudo-first order conditions were maintained for the reaction of reduced auracyanin (100 p~) with oxidized cytochrome c-554 (8 p~) .
There was only sufficient protein available to obtain kinetic data at two concentrations to ensure that the auracyanin:c-554 reaction was second order, and only one ionic strength (96 mM) was studied for this reaction.

RESULTS
Isolation and Biochemical Characterization-Three distinct blue bands eluted from the DEAE-Sephacel column. The elution order was auracyanin A followed by auracyanin B-1 and lastly auracyanin B-2. The B forms ran very close to one another and overlap between the proteins sometimes occurred. The total amount of auracyanin A was greater than the combined amount of auracyanin B-1 and B-2 for each prep. After purification on the hydroxylapatite column, the highest purity ratio (A2w to As%) obtained was 3.54 for auracyanin A, and 3.80 (Am to Am) for B-l and 3.65 The SDS-PAGE apparent monomer molecular masses of the proteins are 14 (A), 18 (B-2), and 22 (B-1) kDa. In the presence of DTT, SDS-PAGE ( Fig. 1) results in only one major band which corresponds to the monomer molecular mass. In the absence of DTT, SDS-PAGE results in two major bands which correspond to the monomer and dimer molecular masses. The gel filtration molecular masses were closest to the estimated SDS-PAGE dimer molecular masses for auracyanin A (23 kDa) and the monomer molecular masses for auracyanin B-1(26 kDa) and B-2 (21 kDa) with or without DTT in the elution buffer. In the presence of 200 mM NaCl (without DTT), however, auracyanin A eluted as a monomer (16 kDa). The molecular masses from sequence analysis are 13.9 (A), 15.6 (B-1), and 14.8 (B-2) kDa. The isoelectric points of the three isolated proteins were found to be 4.0 for auracyanin A, 4.5 for auracyanin B-l, and 3.0 for auracyanin B-2.
Atomic absorption copper analysis gave an extinction coefficient based on copper analysis and absorption at 596 nm for auracyanin A of c5% = 3000 k 100 M" cm", e m = 6400 k 300 M" cm" at 600 nm for B-1, and em = 4500 k 400 M" cm" at 600 nm for B-2. The bicinchoninic acid protein assay coupled with the absorbance at 596 or 600 nm and the estimated monomer molecular mass from amino acid sequence and composition indicated that the auracyanin A sample had 1.01 f 0.09 copper atoms per mol of protein, while B-1 had 0.84 f 0.06 and B-2 had 1.04 & 0.06 copper atoms/mol of protein.
Both auracyanin B-1 and B-2 potentiometric titrations ( Fig. 2) are best fit to the Nernst equation with an n = 1 curve and a midpoint potential of +240 mV with respect to the standard hydrogen electrode. A similar midpoint potential was reported earlier for auracyanin A . The thymol sulfuric acid-stained gel indicated that auracyanin B-l and B-2 were glycoproteins and that auracyanin A was not, and the position of the sugar staining coincided with the staining for protein (data not shown). The neutral sugar analysis using sucrose as a standard, indicated that auracyanin B-1 contained 9.4 f 0.3% (w/w) sugar and auracyanin B-2 contained 3.3 f 0.2%. The auracyanin A and horse heart cytochrome c samples did not react. The a-acid glycoprotein and ovalbumin gave values of 13.0 f 0.4% and 1.6 0.3%, respectively. The experimental standard values were close to reported literature values of 12.0% for a-acid glycoprotein (Bezkorovainy, 1965) and 1.8% for ovalbumin (Fletcher et al., 1963).

Amino Acid Composition and Sequence Determinatwn-
The amino acid compositions of the auracyanin B-forms from both analysis and the proposed sequences are shown in Table   I. The fragments used to determine the protein sequence are listed in Table 11.
The amino acid sequences of auracyanins B-1 and B-2 are shown in Fig. 3. The sequences are identical except for an 8amino acid extension at the N-terminal region in auracyanin B-1. Residues are numbered according to the auracyanin B-1 sequence. The amino acids designated by lower case letters were determined with the help of the fragment amino acid composition due to very small amounts of protein and should be considered tentative.  * Determined from the proposed amino acid sequences.
The cysteine level could not be accurately determined due to an artifact with the same retention time.
The best cleavage results for the auracyanin B forms were usually obtained by using chemical methods at low pH. These included the BNPS-skatole and cyanogen bromide. There was also evidence that under acidic conditions, general acid cleavage occurred at some of the C-terminal residues. This was evidenced by the amino acid composition of the BNPS-Skatole fragments which were missing some of the C-terminal residues. Additionally, several short C-terminal fragments were sequenced after cleavage of the proteins at Met-146 by cyanogen bromide.
The failure of cyanogen bromide to cleave at Met-114/108 is somewhat surprising. An extensive search using both HPLC and SDS-PAGE separations did not find a peptide caused by cleavage at this site. There was also no evidence that the Arg at residue 126 was cleaved by clostripain. These findings would suggest that despite the denaturing conditions used, the protein must maintain a tightly bound structure around the copper binding site.
The lack of an overlapping peptide sequence for residues 109-111, and 144-146 adds some uncertainty to the sequence of the B forms of auracyanin. As mentioned earlier, part of these regions in the sequence (105-110 and 143-145) were based on the amino acid composition of the fragment being sequenced. There was not an amino acid that was an obvious choice for these positions in the sequence, although small amounts of the chosen amino acid were present at the positions that were assigned to them. The use of o-phthaldialdehyde after Pro-102 and -130 reduced the background for only a few cycles, and also resulted in a sharp decrease in yield. Sequence similarity to the auracyanin A form was also used to a lesser extent in assigning the uncertain amino acids.
The N-terminal threonine of auracyanin B-2 (residue 9 of B-1) may be modified in some manner, possibly by glycosylation (see below) In all N-terminal sequences, the amount of threonine at this position was very low in comparison to the amount of threonine at residue 2. It was decided that this residue probably was threonine by comparison to the B-1 sequence and because threonine was clearly present at this position in some samples purified by HPLC using formic acid based solvents.
Spectroscopy-An absorption spectrum typical of blue copper proteins (Fig. 4) is exhibited by all three forms of aura-  * C terminus.

-1 T Q P P A A Q P T T A P A T Q A A N A P G G S N E-2 T T A P A T Q A A N A P G G S N 1 5 10
1s 20

-I S L S L P A N T V V R L D F V N Q N N L G V Q H C Z S L S L P A N T V V R L D F V N Q N N L G V Q H
sa 55 M 65 70

-1 A L F V P P G D l a n a l x W T A M L N A G E S C Z A L F V P P G D I e n a l x W T A M L N A G E S
100 105 110 115 120 cyanin. The individual spectra are very similar, with slight shifts in the absorbance maximum for auracyanin A compared t o the B forms, which are almost identical. The broad absorption maximum for auracyanin A is a t 596 nm and at 600 nm for B-1 and B-2. In addition, there is a small absorption peak near 420 nm and a wide shoulder at 715 nm as well as the 280-nm peak due to aromatic amino acids. The assignment of these peaks to the different copper ligands should be similar t o those found for other blue copper proteins (Solomon et al., 1980;Gewirth and Solomon, 1988). The CD spectra (data not shown) of the three auracyanin forms are generally similar to those of plastocyanin and azurin. Auracyanin A has a CD maximum at 586 nm and minimum at 720 nm, while these occur a t 600 and 733 nm for auracyanin B-1 and B-2. The CD spectrum for auracyanin A was published in Trost et al. (1988).

-1 G S V T F R T P A P G T Y L Y l C T G P G H i p -Z G S V T F R T P A P G T Y L Y I C T G P G H I p
The resonance Raman spectra of the three auracyanins exhibit a set of nine vibrational modes between 330 and 460 cm" (Fig. 5). This set of vibrational frequencies is typical of blue copper proteins where the multiplicity of peaks is ascribed to coupling of the Cu-S(Cys) stretch with Cys ligand deformations (Han et al., 1991). There is an additional vibrational mode at 266 cm" in auracyanin A and 280 cm" in auracyanins B-1 and B-2 (data not shown) that may have Cu-N(His) stretching character. All three auracyanins reveal a similar pattern of resonance Raman intensities with the strongest feature near 420 cm". The intensity pattern appears t o be related to the protein environment of the Cu-cysteinate chromophore and, in this case, is closest to those of azurin and rusticyanin (Han et al., 1991) Exposure of auracyanin A to D20 causes the peaks at 363,386, and the 409 cm-' shoulder to shift by -2, -2, and +1 cm", respectively. This deuterium isotope sensitivity is again typical of blue copper proteins and is indicative of hydrogen bonding between the Cys sulfur ligand and amide NH groups of the protein backbone (Mino et ul., 1987). The X band ESR spectra of the auracyanin forms are shown in Fig. 6. Computer simulations were done for the auracyanin A spectra, and the calculated parameters are compared to those of several other blue copper proteins in Trost et al. (1988). The resolution of three g values in auracyanin A indicates that the geometry about the copper center has rhombic rather than axial distortion. The spectra for B-1 and B-2 auracyanin are not typical of other small type 1 blue copper proteins and could not be accurately computer simulated.
To obtain information as to whether auracyanin functions in photosynthetic electron flow, ESR and optical experiments were carried out on membranes. The light-induced ESR signal in membranes from C. aurantiacus (Fig. 7) is most similar to the isolated auracyanin A ESR spectra. The comparison of ESR signal intensity between nontreated membranes and membranes to which ferricyanide was added indicates that all of the auracyanin in the isolated membranes is initially in the oxidized form. The addition of 0.4 mM ascorbate to these samples reduced approximately 85% of the auracyanin.
A light-induced optical absorbance increase at 600 nm due to oxidation of auracyanin was observed for a membrane sample to which reduced auracyanin was added (data not shown). No increase at 600 nm was seen when auracyanin was not present. Under the same conditions, an increase in absorbance at 554 nm due to the reduction of cytochrome c-554 was observed after illumination ceased when auracyanin was present.
Kinetic Studies-In previous kinetic studies with blue copper proteins, rate constants were measured for reduction of bacterial azurins, nitrite reductase, stellacyanin, and plant and algal plastocyanin by lumiflavin semiquinone (Tollin et al., 1986;Meyer et al., 1987). Reactivities corrected for redox potential differences were similar for all of these proteins with the exception of stellacyanin, which is consistent with known similarities and differences in the three-dimensional structures of the copper proteins (Adman et al., 1978;Norris et al., 1983Norris et al., , 1986Guss and Freeman, 1983;Adman, 1985;Sykes, 1985). In the present study, the second order rate constants for reduction of auracyanin forms A, B-1, and B-2 by lumiflavin semiquinone are 2.2 f 0.1, 2.5 k 0.2, and 3.0 f 0.2 x 10' M" s-l, respectively. These numbers, when corrected for redox potential, are the same as or slightly larger (at most about 1.5-fold) than are those for azurin and plastocyanin, but are not as large as that for stellacyanin (3-fold higher) (Tollin et al., 1986).
The effect of ionic strength on the reduction of auracyanins by fully reduced FMN is shown in Fig. 8. All three proteins showed a minus-minus electrostatic interaction with FMN. Auracyanin form A has an apparent charge at the active site of -1.1 and that for B-1 and B-2 is -1.8 (cf. Tollin et al., 1986 for method of active site charge estimation). These values are similar to or slightly smaller than those for Pseudomonas aeruginosa azurin (Tollin et al., 1986). The most accessible electron transfer site in the latter proteins is commonly referred to as the "hydrophobic patch" (cf. van de Kamp et al., 1990). The observed active site charge in the kinetic experiments is therefore probably due to distant acidic side chains and is related to the net protein charge (-6 on form A auracyanin). The three forms of auracyanin are all acidic proteins and based on the amino acid sequences, it is likely that there is also a hydrophobic electron transfer site, which is influenced by distant negative charge in the same manner as for the other blue copper proteins.
All three forms of auracyanin reacted similarly with purified reaction center cytochrome c-554; typical data are shown in Fig. 9. Reduction of the copper protein alone results in a large rapid absorbance decrease at 600 nm (Fig. 9A); in the presence of cytochrome c-554 there is an initial bleach at 554 nm corresponding to copper reduction, followed by a larger increase above the preflash baseline due to heme reduction and simultaneous copper oxidation (Fig. 9B). Second order rate constants of approximately lo5 M" s" were determined for all of the auracyanin forms. This value is based on protein concentration rather than on heme concentration (there are four hemes in cytochrome c-554 (Freeman and Blankenship, 1991;Dracheva et al., 1991;van Vliet et al., 1991)), inasmuch as auracyanin is expected to react most rapidly with the highest redox potential heme (300 mV) rather than with all four hemes.

Isolation and Biochemical Characterization-The isolation
of more than one blue copper protein from the same organism is not without precedent. In Methylomonas J, two distinct azurins have been isolated and characterized (Ambler and Tobari, 1989). These proteins show a 52% identity to each other when their amino acid sequences are compared. Similarly to the auracyanin forms, the slightly different forms of poplar plastocyanin (Dimitrov et al., 1987) and mung bean blue copper protein (Shichi and Hackett, 1963) also separate differently on DEAE anion-exchange columns. Two different classes of blue copper proteins, amicyanin and pseudoazurin, have been isolated from Pseudomonas AMI (Ambler and Tobari, 1985). In addition to plastocyanin, several higher plants also contain a different blue copper protein in nonphotosynthetic tissue (Adman, 1985). These phytocyanins are often glycoproteins, as are auracyanins B-1 and B-2.
The auracyanin proteins appear to be peripheral membrane proteins because they are released from isolated membranes by salt washing, although it is not yet clear if all of the protein is removed from the membrane by this treatment. If auracyanin functions in a manner analogous to cytochrome c2 in the purple photosynthetic bacteria, then it should be localized on the periplasmic side of the cytoplasmic membrane. The presence of carbohydrate also strongly implies that at least the B forms of auracyanin are located in the periplasm, as bacterial glycoproteins are invariably found there (Wieland, 1988;Fairchild et al., 1991).
All of the type 1 blue copper proteins have a higher midpoint redox potential than the +158 mV for the aqueous Cu2+/Cu1+ couple. The midpoint potential of +240 mV for the auracyanin forms is somewhat low when compared to other blue copper proteins. Most of the blue copper proteins have redox poten- tials close to +300 mV although the range extends from +184 for stellacyanin to +680 mV for rusticyanin. The "tuning" of this potential has been proposed to be due to the position of the copper and the axial ligand(s) (Norris et al., 1986).
The isoelectric points of the isolated proteins would suggest a different order of elution from the DEAE-Sephacel column.
The predicted order of elution would be B-1 first, followed by A and finally B-2 instead of the A, B-1 followed by B-2 order actually seen. This suggests that the additional glycosylation of B-1 might interact with the column or shield some of the charge on the protein.
The difference in apparent molecular mass between the sequence determination, and SDS-PAGE and gel chromatog-raphy molecular masses of auracyanin B-1 and B-2 can be explained by the presence of carbohydrates on these proteins. Auracyanin A, which is not glycosylated, runs close to its sequence-determined molecular mass of 14 kDa. The relatively high percentage of proline in the N terminus of B-1 and B-2 might also affect their mobility in SDS-PAGE and gel chromatography assays. This effect was observed for a group of Photosystem I subunit proteins which contain a proline-rich N terminus including a proline tripeptide (Rousseau and Lagoutte, 1990). These proteins run at a mean apparent molecular mass of 16-17 kDa instead of their calculated values of 9.7-10.8.
Amino Acid Sequence-An interesting aspect of the sequence determination is the strong likelihood that auracyanin B-2 is a processed form of B-1. If this is the case, cleavage must have occurred after a proline (residue 8). The glycosylation in these proteins may play a regulatory role in this processing. The cleavage after a proline residue at the N terminus suggests that a proline-specific protease is present in C. aurantiacus. This opens the question of whether or not the proline at residue 154/146 is the encoded C terminus of these proteins. It is possible that the C terminus extends past this residue in the unprocessed protein.
The site of glycosylation for bacterial N-linked glycoproteins is at a sequence of Asn-X-Thr/Ser with X in this case standing for any amino acid (Lechner and Weiland, 1989). Residues 27-29 (Asn-Glu-Thr) are the only place in the sequence where the auracyanin B forms are likely to have Nlinked carbohydrate groups. It is more likely that the proteins have 0-linked carbohydrates which are found at Thr and Ser residues. Regions rich in Ser, Thr, and Pro have been identified as likely protein domains for 0-glycosylation (Jentoft, 1990). There are several of these residues throughout the protein including the N terminus of both of the B forms of auracyanin. If the N terminus of B-1 is glycosylated, it may play a role in the processing of the protein to form B-2. No attempt was made to identify the site(s) or type(s) of glycosylation in these proteins.
An alignment of the copper binding regions of the auracyanin proteins and other blue copper proteins is shown in Fig.  10. The four copper ligands in the auracyanins are almost certainly two His, one Met, and one Cys. In stellacyanin, which has no methionine, a nearby glutamine (Gln-97), has been proposed as the fourth copper ligand, by comparison to the structure of the cucumber basic blue protein, which is the closest blue protein to stellacyanin in terms of sequence similarity . Auracyanin A is unique among blue proteins in having both a methionine and glutamine at this point in the sequence.
Auracyanin A shows an overall identity of 38% (identical amino acid matches) when aligned with the B forms (Mc-Manus, 1990).' The similarity around the copper binding region, however, is quite high. This may explain why the different forms of auracyanin have so many similar properties.
The overall sequence identity of the auracyanins to other small blue proteins ranges from a maximum of 30% for some plastocyanins to 14% for the cucumber basic blue protein (data not shown). The auracyanins occupy an evolutionary position approximately equidistant between the plastocyanins and the azurins. A more complete comparative analysis of the sequences of all the blue proteins, including both types of auracyanins is presented in McManus (1990).' Properties of the Blue Copper Site-The resonance enhancement of blue copper protein Raman spectra is derived from the (Cys)S + Cu'+ charge transfer transition (Woodruff et al., 1988). The extensive coupling of the Cu-S stretch with Cys ligand deformation modes is facilitated by the coplanarity of the Cu-S&-C,-N atoms in the copper cysteinate moiety (Han et al., 1991). The observation of the expected nine vibrational modes between 330 and 460 cm" in the auracyanins indicates that the conserved coplanar cysteine ligand conformation is also present in these three proteins. In contrast, Raman intensities are remarkably variable among the different blue copper proteins, and this variability appears to correlate with the size of the loop connecting the Cys and His ligands (Han et al., 1991). The similar spectral patterns with the most intense feature at 410-420 cm" for auracyanins (A, B-1, B-2), azurins, and rusticyanin correlates well with the n + 5 spacing between the Cys(n) and His ligands in all of these proteins. The identical Raman spectra for auracyanins B-1 and B-2 indicates identical protein structures in this region of the blue copper site, as expected from their 100% sequence identity in the copper binding domain. The small differences in Raman frequencies and intensities between the A and B forms are due to small structural perturbations, as would be expected from the presence of only a 50% sequence identity in the copper binding domains. Similar Raman spectral differences are observed for two species of plastocyanin that differ by only 11 amino acids, all of which are conservative changes and none of which are close to the copper site (Han et al., 1991).
The unusual hyperfine splitting seen in the ESR spectra of the B-1 and B-2 forms is not characteristic of either the type 1 or type 2 copper centers. They are somewhat similar to the ESR spectra of the purple form of nitrous oxide reductase from Pseudomonas stutzeri (Jin et al., 1989). This is intriguing since nitrous oxide reductase is a 140,000-kDa multicopper protein composed of two identical subunits. One proposal to explain its unusual hyperfine splitting pattern is that an overlap exists between two type 1 copper centers with different gJJ values (Jin et al., 1989). A similar proposal has been made for the unusual ESR signal observed in the CUA center of cytochrome oxidase (Kroneck et al., 1990), although in that system it is not clear whether or not sufficient copper is present for interacting centers (Chan and Li, 1990). However, multiple copper interactions seem to be unlikely in the auracyanin B forms since both copper analysis and the amino acid sequence suggest that there is only 1 copper/monomer. Also, only one set of resonance Raman peaks is observed for each protein. Under some conditions, the auracyanins appear to dimerize in solution, which could conceivably give rise to an interacting pair of copper centers, although this is much more pronounced for the A form than the B forms. Additional experiments are in progress to determine if the ESR spectra of the B forms are correlated with the aggregation state of the proteins. Kinetic Studies-All three forms of auracyanin reacted similarly with lumiflavin semiquinone and were a little more reactive than was previously observed for azurin and plastocyanin (Tollin et al., 1986), suggesting somewhat greater exposure of the copper site to solvent. All three forms showed an apparent -1 to -2 charge at the site of reduction, which is consistent with reaction at the "hydrophobic patch," i.e. the region at which the copper is nearest the surface. This is similar to previous observations with azurin and plastocyanin, for which the charge at the site of reduction had the same sign as the net protein charge but was of smaller magnitude (Tollin et al., 1986). Three-dimensional structures indicate that there are no charged surface amino acid residues on the face of the copper proteins where the copper is nearest to the surface; hence the term "hydrophobic p a t c h (Adman et al., 1978;Norris et al., 1983;Guss and Freeman, 1983;Petratos et al., 1987;Guss et al., 1988;Collyer et al., 1990). Based on the amino acid sequence of auracyanin, it is expected that the immediate surface environment of the copper will also be hydrophobic and that more distant charged residues will be responsible for the ionic strength effect.
The reactivity of auracyanin with the tetraheme reaction center-associated cytochrome c-554 is not particularly high (second order rate constant of lo5 M" s-l at 100 mM ionic strength). Unfortunately, there was too little protein available to determine the effect of ionic strength on the reaction kinetics, and it is possible that the rate constant may be larger at higher or lower ionic strength. Measurement of the kinetics of reduction of cytochrome c-554 by free flavins showed that the highest redox potential heme 3 (300 mV) was most reactive and has a negative charge at the site of reduction (Meyer et al., 1989). If auracyanin also reacts with the highest potential heme, then a minus-minus interaction would be expected and the rate constant would be even smaller at lower ionic strength than that which was measured. The reaction of Pseudomonas azurin with Rps. viridis photosynthetic reaction center3 appears to be about an order of magnitude slower than that between C. auruntincus auracyanin and cytochrome c-554. However, these reactions are not exactly comparable, because the azurin (327 mV) reaction was with the second lowest potential heme 2 (300 mV) rather than with the highest potential heme 3 (380 mV). Furthermore, the redox potential difference in this case was unfavorable. Another complication is that the C. aurantiacus cytochrome c-554 was free in solution, whereas in the Rps. viridis experiment, the cytochrome was bound to the reaction center. The reaction of auracyanin with c-554 may have been with the same site on the cytochrome as that which interacts with the reaction center in the complex. Thus, auracyanin may be much less reactive with the c-554 when it is bound to the reaction center than in solution, and thus may be comparable to that of azurin with reaction center cytochrome in Rps. viridis. More kinetic experiments are needed to determine the reactivity of auracyanin with electron transfer components.
Proposed Function-The continuous illumination experiments suggest that auracyanin is oxidized by cytochrome c-554 during photosynthetic electron transport. Auracyanin '' Meyer, T. E., Cusanovich, M. A,, and Tollin, G., unpublished data. oxidation could occur as it donates an electron to the reaction center-bound cytochrome c-554. The photoinduced ESR signal observed in isolated C. aurantiacus membranes is similar to that of isolated auracyanin A. Part of the noise in the spectrum could be due to the signal from the B forms which are present in much lower quantities. This result suggests that auracyanin is involved in photosynthetic electron transfer, although kinetic studies with higher time resolution are needed to establish the reaction pathway. Photoinduced plastocyanin ESR signals were observed in intact algal cells (Visser et al., 1974) and spinach chloroplasts (Malkin and Bearden, 1973). The in vitro kinetics of interaction of auracyanin with cytochrome c-554 also suggests that they could be reaction partners in vivo.
The photosynthetic electron transfer scheme in C. aurum tiucus is not yet completely understood. It appears to be similar to the purple photosynthetic bacteria in that it has a pheophytin-quinone photosynthetic reaction center (Bruce et al., 1982;Blankenship, 1985;Amesz, 1987;Kirmaier and Holten, 1987). The cytochrome bc1 complex of C. aurantiacus has not yet been isolated. Evidence for this complex was found by Zanonni and Ingledew (1985) who identified appropriate midpoint potentials for both band C-type cytochromes and an ESR signal for a photooxidizable Rieske iron sulfur center. The lack of soluble C-type cytochromes in C. aurantiacus (Bartsch, 1978;Wynn et al., 1987;Freeman and Blankenship, 1990) suggests a void in the probable electron transfer scheme which may be filled by auracyanin A.
A proposed electron transfer scheme in C. aurantiacus is shown in Fig. 11. The auracyanins have a midpoint redox potential of +240 mV which makes it thermodynamically possible for them to donate electrons directly to the oxidized reaction center (+340 mV) or through the membrane-bound cytochrome c-554 (+260 mV). The role of auracyanin A has been proposed to be in photosynthetic electron transfer, replacing the soluble cytochrome cp found in purple photosynthetic bacteria . The continuous illumination experiment and photoinduced ESR signal support this proposal. The kinetic evidence suggests that all three forms of auracyanin may be reactive with the bound reaction center cytochrome c-5 high. Because the auracyanin blue copper proteins appear to function in electron transfer in an anoxygenic photosynthetic bacterium, they are likely to be similar to the plastocyanins which transfer electrons between the cytochrome bGf complex and the reaction center of Photosystem I of some cyanobacteria and almost all eukaryotic photosynthetic organisms. The amino acid sequences of the auracyanins are approximately equidistant between those of plastocyanins and azurins2 Their distinct biochemical and physical properties, amino acid sequences, and unique existence in a single-photosystemcontaining bacterium, however, clearly establishes the auracyanins as a novel class of blue copper proteins.