Protein engineering for the elucidation of the mechanism of electron transfer in redox proteins.
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Anabaena↗
Biomedical subjects
G Tollin
Publications and source records attributed to G Tollin.
Laser flash-induced photoreduction of photosynthetic ferredoxins and flavodoxin by 5-deazariboflavin and by a viologen analogue.
Laser flash photolysis has been used to compare the kinetics of reduction of ferredoxin isoforms from the green alga Monoraphidium braunii, and the ferredoxin and flavodoxin from the cyanobacterium Anabaena PCC 7119, by 5-deazariboflavin semiquinone (dRfH.) and the viologen analogue 1,1'-propylene-2,2'-bipyridyl (PDQ.+). Similar ionic strength-independent second-order rate constants (1.4 x 10(8) M-1 s-1) were obtained for the reduction of both algal ferredoxin isoforms by dRfH.. For the reduction of oxidized flavodoxin by dRfH., a more complex behavior was observed, with a second-order rate constant for dRfH. decay of 1.8 x 10(8) M-1 s-1, and a first-order (i.e. protein concentration independent) rate constant of 450 s-1, that probably corresponds to the protonation of the FMN semiquinone cofactor, which occurs subsequent to electron transfer. A value of 5 x 10(7) M-1 s-1 was obtained for the second-order rate constant of flavodoxin semiquinone reduction by dRfH.. The reduction of ferredoxins and flavodoxin semiquinone by PDQ.+ showed nonlinear protein concentration dependencies, consistent with a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer. A negative ionic strength effect on the kinetic constants was obtained, indicating the existence of attractive electrostatic interactions during electron transfer. With all the ferredoxins the k infinity values (rate constants extrapolated to infinite ionic strength) for the second-order step of the reduction process (complex formation) are smaller than previously reported for spinach ferredoxin, although Anabaena ferredoxin is somewhat more reactive than are the algal ferredoxins with the viologen.(ABSTRACT TRUNCATED AT 250 WORDS)
Anabaena↗
Oxidative turnover increases the rate constant and extent of intramolecular electron transfer in the multicopper enzymes, ascorbate oxidase and laccase.
Using laser flash photolysis of lumiflavin/EDTA solutions containing ascorbate oxidase, we find that the rate constant for intramolecular electron transfer varies from one enzyme preparation to another and is generally a more sensitive measure of the state of the active site than are steady-state assays. Thus, type I copper is initially reduced in a second-order reaction followed by first-order reoxidation by the type II-III trinuclear copper center. The observed rate constant for this intramolecular process in presumably native enzyme is 160 s-1 at pH 7, whereas an enzyme preparation which had less than 20% activity had a rate constant of 2.6 s-1. Other samples of relatively active enzyme showed biphasic intramolecular kinetics intermediate between the above values. The inactive enzyme sample could be reactivated by dialysis against ascorbate or by treatment with ferricyanide, resulting in a corresponding increase in the intramolecular rate constant for type I copper reoxidation to a value comparable to that of native enzyme. Using this same methodology, we have determined that the type I copper in Japanese lacquer tree laccase is reoxidized by the type II-III trinuclear copper center in a first-order (intramolecular) process with rate constants of 1 s-1 at pH 7.0 and 4.9 s-1 at pH 6.0, values which are approximately two orders of magnitude smaller than for ascorbate oxidase. The intramolecular rate constant and enzyme activity for laccase also increased, but only by a factor of 2-6, when the enzyme was treated with ascorbate or ferricyanide, respectively. We further found that intramolecular electron transfer in laccase was completely inhibited by fluoride ion, in contrast to ascorbate oxidase which is unaffected by this ion. These differences in behavior for these two very similar enzymes are rather remarkable, when it is considered that the distance between copper atoms is constrained by the location of the protein-derived copper ligands in the three-dimensional structure, and that the redox potentials of the enzymes are similar. Our results may be interpreted in terms of an interconversion between active and inactive enzyme in which there is a rearrangement of the type II-III trinuclear copper center, resulting in a lowering of the redox potential and a block in electron transfer. Turnover restores the active enzyme conformation.(ABSTRACT TRUNCATED AT 400 WORDS)
Ascorbate Oxidase↗
Electron transfer in sulfite oxidase: effects of pH and anions on transient kinetics.
Intramolecular electron transfer (ET) rates in sulfite oxidase (SO) were measured using flavin semiquinone reductants [5-deazariboflavin (dRFH.) and lumiflavin (LFH.)] generated by laser flash photolysis. Rapid bimolecular reduction of the heme by the dRF semiquinone occurred (k = 4 x 10(8) M-1 s-1 at pH 6; 1 x 10(8) M-1 s-1 at pH 9), followed by heme Fe(II) reoxidation due to intramolecular electron transfer to Mo(VI). Flash-induced difference spectra indicated that only spectral processes due to reduction and oxidation of the b-type heme prosthetic group were observed, with no detectable spectral contribution from the Mo cofactor. The extent of reoxidation decreased greatly from pH 6 to 9 (50% to 3%), as expected from the shifts in the redox potentials of the heme and Mo cofactor with pH, consistent with an electron transfer equilibrium between the two redox centers. The observed rate constant for the Fe(II) to Mo(VI) electron transfer decreased from 1650 s-1 at pH 6 to 60 s-1 at pH 9 and showed a maximum of 2400 s-1 at pH 7. Increases in salt concentration greatly decreased intramolecular ET rate constants (direct reduction by flavin semiquinone was unchanged), due to the binding of anions. Titration with the sodium salts of Cl-, SO4(2-), and H2PO4-/HPO4(2-) resulted in decreases in rate constants of intramolecular ET from 1500 s-1 to < 100 s-1 at pH 6 and 7. Similar dissociation constants were measured for the binding of these anions by flash photolysis and by steady-state enzyme kinetics using the inhibition of the sulfite/cytochrome c assay reaction for sulfite oxidase. A mechanism is proposed in which anion binding to the enzyme inhibits the rate of intramolecular electron transfer.
Animals↗
Transient proton uptake and release is associated with the photocycle of the photoactive yellow protein from the purple phototrophic bacterium Ectothiorhodospira halophila.
Upon excitation by a laser flash at 445 nm, the photoactive yellow protein (PYP) undergoes a bleach and red-shift occurring in less than 10 ns, undergoes a further bleach in approximately 200 microseconds, and then recolors in approximately 200 ms. A conformational change occurs during photobleaching which exposes a hydrophobic site. We have now shown that this photocycle also involves a net uptake of one proton during formation of the fully bleached second intermediate, followed by an equivalent proton release upon return of PYP to the ground state. Proton uptake lags slightly behind PYP bleaching and is first-order, indicating that the protein conformational change occurs in two steps. The results suggest that a basic residue which is normally buried in the protein interior is transiently exposed to solvent during the PYP photocycle and, as a consequence, undergoes a change in pK. On the basis of the crystal structure of PYP, we propose that this basic residue is lysine 111.
Bacterial Proteins↗
Direct measurement of cyclic current-voltage responses of integral membrane proteins at a self-assembled lipid-bilayer-modified electrode: cytochrome f and cytochrome c oxidase.
Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.
Conductometry↗
Kinetics of photooxidation of soluble cytochromes, HiPIP, and azurin by the photosynthetic reaction center of the purple phototrophic bacterium Rhodopseudomonas viridis.
The photosynthetic reaction center of Rhodopseudomonas viridis contains a bound tetraheme cytochrome c subunit which is the primary electron donor to the photooxidized special pair bacteriochlorophyll. We have tested a variety of soluble electron-transfer proteins for their ability to serve as secondary electron donors to the bacteriochlorophyll via the bound cytochrome by measuring the kinetics of reaction center heme reduction following photooxidation by a laser flash, as a function of soluble protein concentration and ionic strength. All of the soluble redox proteins utilized appear to interact with a negatively charged region on the reaction center and to transfer electrons to the 300-mV heme c-556 of the bound cytochrome. Rps. viridis cytochrome c2 was the best electron donor among those proteins tested, with a second-order rate constant extrapolated to infinite ionic strength of 1.2 x 10(6) M-1 s-1, which is two orders of magnitude larger than that of horse cytochrome c. Rps. viridis cytochrome c2 apparently binds to the reaction center at low ionic strength, as evidenced by a nonlinear dependence of kobs on protein concentration. The limiting first-order electron-transfer rate constant at 6 mM ionic strength is approximately 1300 s-1. Horse cytochrome c and the reaction center also form a complex, with a limiting first-order rate constant for electron transfer which is 5 times smaller than for cytochrome c2. Other cytochromes c2 are intermediate in reactivity. More distantly related cytochromes, HiPIP, and azurin are relatively poor electron donors under the conditions of assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Azurin↗
Transient kinetics of electron transfer from a variety of c-type cytochromes to plastocyanin.
Plastocyanin (PC) and its physiological reaction partner cytochrome (cyt) f form a complex which is electrostatically stabilized by interactions between complementary localized charges. We have measured the kinetics of intracomplex electron transfer between several reduced cytochromes and PC using laser flash photolysis. With spinach cyt f and spinach PC, we obtain first-order rate constants, kforward = 2780 s-1 and kreverse = 1050 s-1, for the reversible reaction and a complex dissociation constant of about 23 microM at an ionic strength (I) of 5 mM. The observed rate constant increases by a factor of 2 between I = 5 and 40 mM and then decreases monotonically at higher ionic strengths. This indicates that the complex is not completely dissociated until I = 150 mM and that the proteins within the electrostatically most stable complex are not optimally oriented for electron transfer. Similar results were obtained with turnip cyt f and spinach PC, although in this case intracomplex electron transfer is about 4 times as fast. Horse cyt c also forms an electrostatically stabilized complex with PC, and yields a limiting rate constant for intracomplex electron transfer (1750 s-1) and a dissociation constant (10 microM) comparable to those for spinach cyt f. The ionic strength dependence shows that the complex is more readily dissociated (complete at I = 25 mM) than is that of cyt f and that rearrangement is not required for optimal electron transfer. Addition of polylysine results in 10-fold inhibition of the rate of electron transfer. Pseudomonas cyt c-551 is an acidic cytochrome which does not form a complex with PC.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Proteins↗
A comparative laser-flash absorption spectroscopy study of Anabaena PCC 7119 plastocyanin and cytochrome c6 photooxidation by photosystem I particles.
Laser-flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c6 and plastocyanin, isolated from Anabaena PCC 7119, to oxidized P700 in photosystem-I particles isolated from the same cyanobacterium and from spinach. For all metalloproteins and photosystems, the observed rate constant has a non-linear protein-concentration dependence, thus suggesting complex formation preceding electron transfer. Plastocyanin and cytochrome c6 have similar association constants for complex formation with spinach photosystem I, but the copper protein exhibits a higher intracomplex-electron-transfer rate constant (twofold). With Anabaena photosystem I, the two redox proteins are more effective with respect to both complex formation (5-10-fold) and electron transfer (1.5-4-fold) than with the spinach photosystem. In all cases, the observed rate constants for electron-transfer monotonically decrease with increasing NaCl or MgCl2 concentration. This is interpreted in terms of the involvement of attractive electrostatic interactions, which result in the initial collision complex having the most productive orientation for the electron transfer process, without a requirement for further reorientation. The magnitude of the response to MgCl2 suggests the occurrence of specific ion effects as well. In the absence of added salts, the reduction rate of oxidized P700 increases with pH from approximately 6 to 8, but decreases slightly at pH 8.5.
Anabaena↗
Effects of surface amino acid replacements in cytochrome c peroxidase on intracomplex electron transfer from cytochrome c.
Transient absorption techniques were used to measure the intracomplex electron transfer rates between four recombinant yeast cytochrome c peroxidases and iso-1 cytochrome c (cytc). The binding affinities and catalytic activities with cytc were previously examined [Corin et al. (1991) Biochemistry 30, 11585]. The four include a wild-type peroxidase (ECcP) and three others, each of which has one surface aspartic acid converted to lysine at position 37, 79, or 217. These sites have been suggested to be within or proximal to the recognition site for cytc. These mutants conduct electron transfer with cytc but differ with respect to the ionic strength profiles of their limiting rate constants. At pH and mu = 114 mM, ECcP and D217K show similar limiting rate constants for electron transfer with cytc, k(lim), of ca. 2000 s-1. In the same peroxidase concentration range, the D37K mutant exhibits a k(obs) of ca. 100 s-1. Instability of the compound I form of D79K prevented a complete study of the intracomplex kinetics of this mutant by this technique. At pH 6 and low ionic strength (8 mM), D37K exhibits a dramatic increase in k(obs) to ca. 800 s-1 while the other two recombinants show a marked decrease to values < 150 s-1. D37K displays much lower affinity for cytc than do the other peroxidases at higher ionic strengths [Hake et al. (1992) J. Am. Chem. Soc. 114, 5442], thus preventing adequate complexation necessary for efficient electron transfer. Variations in binding affinity do not explain the more subtle ionic strength kinetic profile observed for D217K.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex.
Rate constants for electron transfer in the complex between recombinant rat mitochondrial outer membrane cytochrome b5 or the tryptic fragment of bovine liver cytochrome b5 and horse mitochondrial cytochrome c were measured by laser flash photolysis of 5-deazariboflavin-EDTA solutions. When an excess of cytochrome b5 was titrated with increasing amounts of cytochrome c at low ionic strength and electron transfer was initiated by a laser flash, both proteins were rapidly reduced by deazariboflavin semiquinone. The initial photoreduction was followed by a slower second-order reduction of b5 complexed oxidized cytochrome c by free reduced cytochrome b5. At an 8:1 ratio of cytochromes b5 to c, the pseudo-first-order rate constant for reduction of complexed cytochrome c increased 3-5-fold between ionic strengths of 5 and 40 mM, and then dropped precipitously at higher ionic strengths. The ionic strength dependent increase in rate constant is likely to be due to relief of steric hindrance via rearrangement of cytochrome c in the complex. The reaction rate showed no sign of saturation at any ionic strength, indicating a first-order rate constant greater than 10(4) s-1 within a transient ternary protein complex; i.e., interprotein electron transfer approaches the largest values previously reported for the stable binary protein complex (approximately 4 x 10(5) s-1). Our results emphasize the flexibility of electron-transfer protein complexes, which had previously been modeled in a single conformation with specific salt bridges. It appears that a variety of orientations can exist within such protein-protein complexes and that the population of conformations changes with ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Purification and properties of an unusual membrane-derived cytochrome b-561 from the purple phototrophic bacterium Rhodobacter capsulatus, which is structurally related to the bacteriochlorophyll-binding protein, LHII beta.
An abundant cytochrome b-561 was solubilized from Rhodobacter capsulatus membranes by successive treatments with perchlorate and butanol/water. Neither procedure was effective alone although they could be combined into a single step. Once solubilized, cytochrome b-561 was purified by standard chromatographic procedures used for water-soluble proteins without addition of butanol or detergents. Cytochrome b-561 appears to be highly acidic, it has a size greater than about 1000 kDa as isolated, and the subunit size measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is less than 8 kDa. The redox potential measured by cyclic voltammetry is -65 mV at pH 7. The N-terminal amino acid sequence is identical to that of the Rb. capsulatus LHII beta light-harvesting bacteriochlorophyll binding protein subunit which has only 48 amino acid residues, and the mass, determined by mass spectroscopy, is identical to that of LHII beta. There is but one heme per two to three peptide chains of 5 kDa, which suggests that the two extraplanar ligands to the heme are on separate subunits. There is strong exciton splitting in the circular dichroism spectrum in the Soret region indicative of heme-heme interaction. The helix content based on far-uv CD is 41%. Together, these properties of cytochrome b-561 are very similar to those of isolated LHII alpha beta bacteriochlorophyll-protein complexes.
Amino Acid Sequence↗
Use of laser flash photolysis time-resolved spectrophotometry to investigate interprotein and intraprotein electron transfer mechanisms.
A description is given of the methodology developed in our laboratory for the application of laser flash photolysis to the elucidation of the kinetics and mechanism of electron transfer processes which occur intermolecularly between two protein molecules within a collisional complex, or intramolecularly between two redox centers within a single multisubunit or multidomain protein. This involves the use of flavin analogs, excited to their lowest triplet state by a laser flash, to initiate electron transfer, either by oxidation of a sacrificial donor followed by redox protein reduction via the flavin semiquinone, or by direct oxidation of a reduced redox protein by the flavin triplet. Time-resolved spectrophotometry is used to follow the course of the sequence of electron transfer events initiated by the laser flash. The application of this methodology to the following systems is described: cytochrome c/cytochrome c peroxidase; ferredoxin/ferredoxin NADP+ reductase; cytochrome c/plastocyanin; flavocytochrome b2; and sulfite oxidase.
Animals↗
Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions.
The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.
Anabaena↗
A laser flash absorption spectroscopy study of Anabaena sp. PCC 7119 flavodoxin photoreduction by photosystem I particles from spinach.
Electron transfer from P700 in photosystem I (PSI) particles from spinach to Anabaena sp. PCC 7119 flavodoxin has been studied using laser flash absorption spectroscopy. A non-linear protein concentration dependence of the rate constants was obtained, suggesting a two-step mechanism involving complex formation (k = 3.6 x 10(7) M-1.s-1) followed by intracomplex electron transfer (k = 270 s-1). The observed rate constants had a biphasic dependence on the concentrations of NaCl or MgCl2, with maximum values in the 40-80 mM range for NaCl and 4-12 mM for MgCl2. To our knowledge, this is the first time that the kinetics of PSI-dependent flavodoxin photoreduction have been determined.
Cyanobacteria↗
Direct electrochemistry of thioredoxins and glutathione at a lipid bilayer-modified electrode.
By using direct electrochemical analysis we have established that the reduction of Escherichia coli thioredoxin (EcT), T4 thioredoxin (T4T), and glutathione (GSSG) occurs at a self-assembled lipid bilayer-modified gold electrode via two separate one-electron processes. The first electron transfer has half-wave potentials of -0.05 +/- 0.01, -0.07 +/- 0.01, and -0.06 +/- 0.01 V, whereas the second one has values of -0.48 +/- 0.01, -0.39 +/- 0.01, and -0.45 +/- 0.01 V, for EcT, T4T, and GSSG, respectively. The scan-rate dependence of the cyclic voltammetry indicates, for both waves, that the process of electron transfer is dominated by a bulk diffusion of free species to and from the electrode, and that strongly adsorbed species do not significantly contribute at the scan rates used. The voltage separation of the peak currents indicates a quasi-reversible electron transfer process with an electrochemical rate constant which is larger for the second (lower potential) electron than for the first one. Using the above half-wave potentials of the one-electron steps, one can calculate a thermodynamic half-wave potential for the two-electron reduction processes. The values of these potentials are -0.265, -0.23, and -0.25 V for EcT, T4T, and GSSG, respectively. These are in excellent agreement with literature values obtained from equilibrium measurements of enzyme-catalyzed reactions involving these species. It is quite clear from these results that lipid bilayer-modified electrodes provide a biocompatible and direct means of efficiently carrying out electrochemical reactions with sulfur-based redox systems, as we have previously shown to be the case with metalloproteins.
Electrochemistry↗
Direct electrochemistry of spinach plastocyanin at a lipid bilayer-modified electrode: cyclic voltammetry as a probe of membrane-protein interactions.
The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.
Electrochemistry↗
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-1, 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, 7858-7863) 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 electrophoresis 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 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.
Amino Acid Sequence↗