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G Tollin

Publications and source records attributed to G Tollin.

At least 37 records · Page 2Linked to original sources
PubMed1999

Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in </=3 ps, and I(0)(double dagger), which is formed in 220 ps, as well as stimulated emission from the PYP excited state. In the present study, kinetic measurements at two excitation wavelengths (395 nm and 460 nm) on either side of the PYP absorption maximum (446 nm) were undertaken using 100-fs pump and probe pulses. Global analysis over a range of probe wavelengths yielded time constants of 1.9 ps for the photochemical formation of the I(0) intermediate via the PYP excited state, and 3.4 ps for the repopulation of the ground state from the excited state. In addition to these pathways, 395 nm excitation also initiated an alternative route for PYP excitation and photochemistry, presumably involving a different excited electronic state of the chromophore. No photochemical intermediates formed before I(0) were observed. Based on these data, a quantum yield of 0.5-0.6 for I(0) formation was determined. The structural and mechanistic aspects of these results are discussed.

Bacteria↗
PubMed1999

Plasmon resonance spectroscopy: probing molecular interactions within membranes.

Surface plasmon resonance (SPR) has become a popular method for investigating biomolecular interactions. A new variant of this technique, coupled plasmon-waveguide resonance (CPWR) spectroscopy, allows the characterization of anisotropic biological membranes. Plasmon resonance can therefore be used to study the molecular events involved in a wide variety of membrane processes, including energy conversion and signal transduction.

Animals↗
PubMed1999

Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NADP+ reductase: site-specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials.

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein-protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein-protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.

Anabaena↗
PubMed1998

Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal NADP+ binding and electron transfer.

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg100 and Arg264 may be directly involved in the proper NADP+/NADPH orientation for an efficient electron-transfer reaction. Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119 FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP+ and to ferredoxin/flavodoxin. Arg100 has been replaced with an alanine, which removes the positive charge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutation has been made at Arg264 by replacing it with a glutamic acid. Results with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Both mutants have been kinetically characterized by steady-state as well as fast transient kinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved. The results reported herein reveal important conceptual information about the interaction of FNR with its substrates. A critical role is confirmed for the long, positively charged side chain of Arg100. Studies on the Arg264Glu FNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or for nicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behavior in its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin.

Alanine↗
PubMed1998

Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer.

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH. However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position. These kinetic parameters also suggest that it is complex formation that is affected by mutation. Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions. This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners. The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.

Amino Acid Sequence↗
PubMed1998

New insights into the photocycle of Ectothiorhodospira halophila photoactive yellow protein: photorecovery of the long-lived photobleached intermediate in the Met100Ala mutant.

There are previously two known intermediates (I1 and I2) in the room-temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The three-dimensional structures of ground-state PYP and of I2 have shown that light-induced conformational changes are localized to the active site. Previous site-specific mutagenesis studies of PYP in our laboratories have characterized two active site mutants (Glu46Gln and Arg52Ala). We now report the construction and characterization of a mutant at a third active site position (Met100Ala) in order to establish the role of this residue in the photocycle. Met100Ala PYP has an absorption spectrum which is very similar to wild-type (WT) PYP, but exhibits very different kinetic properties. At pH 7.0, the light-induced bleaching reaction (I2 formation) has a half-life <1 microseconds and the recovery in the dark has a half-life of 5.5 min, as compared with half-lives of 100 microseconds and 140 ms for the same reactions in WT PYP. The slow rate of recovery from I2 for Met100Ala results in the accumulation of the bleached intermediate even under room light illumination. These results are qualitatively similar to what has been observed with the Arg52Ala mutant of PYP, and with WT PYP in the presence of alcohols or urea, and suggest that Met100 acts to stabilize the ground state of the protein. The midpoint for guanidine denaturation confirms this. The slow recovery of I2 in the Met100Ala mutant has allowed us to obtain direct evidence that this intermediate species is also photoactive and can be returned to the ground state by a 365 nm laser flash, with kinetics (half-life = 160 microseconds; k = 6300 s-1) which are 6 orders of magnitude faster than dark recovery. This implies that chromophore reisomerization limits the rate of conversion of I2 to the ground state in PYP. Met100 is in van der Waals contact with the chromophore in the I2 state, and we suggest that the sulfur atom catalyzes cis-trans isomerization in WT PYP.

Alanine↗
PubMed1998

An electrochemical, kinetic, and spectroscopic characterization of [2Fe-2S] vegetative and heterocyst ferredoxins from Anabaena 7120 with mutations in the cluster binding loop.

Residues within the cluster binding loops of plant-type [2Fe-2S] ferredoxins are highly conserved and serve to structurally stabilize this unique region of the protein. We have investigated the influence of these residues on the thermodynamic reduction potentials and rate constants of electron transfer to ferredoxin:NADP+ reductase (FNR) by characterizing various single and multiple site-specific mutants of both the vegetative (VFd) and the heterocyst (HFd) [2Fe-2S] ferredoxins from Anabaena. Incorporation of residues from one isoform into the polypeptide backbone of the other created hybrid mutants whose reduction potentials either were not significantly altered or were shifted, but did not reconcile the 33-mV potential difference between VFd and HFd. The reduction potential of VFd appears relatively insensitive to mutations in the binding loop, excepting nonconservative variations at position 78 (T78A/I) which resulted in approximately 40- to 50-mV positive shifts compared to wild type. These perturbations may be linked to the role of the T78 side chain in stabilizing an ordered water channel between the iron-sulfur cluster and the surface of the wild-type protein. While no thermodynamic barrier to electron transfer to FNR is created by these potential shifts, the electron-transfer reactivities of mutants T78A/I (as well as T48A which has a wild-type-like potential) are reduced to approximately 55-75% that of wild type. These studies suggest that residues 48 and 78 are involved in the pathway of electron transfer between VFd and FNR and/or that mutations at these positions induce a unique, but unproductive orientation of the two proteins within the protein-protein complex.

Anabaena↗
PubMed1998

A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.

Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.

Amino Acid Sequence↗
PubMed1998

Ligand binding and covalent structure of an oxygen-binding heme protein from Rhodobacter sphaeroides, a representative of a new structural family of c-type cytochromes.

The amino acid sequence of an oxygen-binding heme protein (SHP) from Rhodobacter sphaeroides has been determined. The cysteines, which bind the single heme group in the 112-residue protein, are located at positions 43 and 46. SHP is similar in size to the large membrane-bound form of the class I cytochrome c5 of Azotobacter vinelandii (116 residues) and in the location of the heme binding site at positions 48 and 51. Two extra cysteines in SHP (residues 89 and 97) are located in positions similar to those of cytochrome c5 (residues 98 and 101) and form a disulfide bridge in both proteins. In total, four regions of alpha-helix are predicted, covering 46% of the protein, which is comparable to that in other small cytochromes. SHP is thus distantly related to small class I c-type cytochromes but is representative of a distinct family by virtue of its high-spin nature, the lack of a strong sixth ligand, and its capacity to bind oxygen. Potentially, the most important characteristic of SHP is its ability to transiently bind oxygen during autoxidation, which occurs with a half-life of 3 min with a 4-fold excess of O2. SHP also binds carbon monoxide, azide, and cyanide. The kinetics of reduction by free flavins indicate that SHP is less reactive than other class I cytochromes c and that the heme is less accessible to solvent. There is localized positive charge (+3) at the site of reduction of SHP, although the overall protein charge is -2. This may account in part for the ability of SHP to bind anions.

Amino Acid Sequence↗
PubMed1998

Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array.

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

Calorimetry, Differential Scanning↗
PubMed1998

Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119.

The crystal structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.

Anabaena↗
PubMed1998

The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase.

Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the Kms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.

Amino Acid Oxidoreductases↗
PubMed1998

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.

Biophysical Phenomena↗
PubMed1998

Coupled plasmon-waveguide resonance spectroscopy studies of the cytochrome b6f/plastocyanin system in supported lipid bilayer membranes.

The incorporation of cytochrome (cyt) b6f into a solid-supported planar egg phosphatidylcholine (PC) bilayer membrane and complex formation with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance (CPWR) spectroscopy, developed in our laboratory. CPWR combines greatly enhanced sensitivity and spectral resolution with direct measurement of anisotropies in refractive index and optical extinction coefficient, and can therefore probe structural properties of lipid-protein and protein-protein interactions. Cyt b6f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characterized by an increase in total proteolipid mass without significant changes in the molecular order of the system, as demonstrated by shifts of the resonance position to larger incident angles without changing the refractive index anisotropy. The second stage, occurring at higher protein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the resonance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a proteolipid system in three different ways. First, the addition of plastocyanin before the second stage of b6f incorporation begins results in complex formation between the two proteins with a KD of approximately 10 microM and induces structural changes in the membrane that are similar to those occurring during the second stage of complex incorporation. The addition of larger amounts of plastocyanin under these conditions leads to nonspecific binding to the lipid phase with a KD of approximately 180 microM. Finally, the addition of plastocyanin after the completion of the second phase of b6f incorporation results in tighter binding between the two proteins (KD approximately 1 microM). Quantitation of the binding stoichiometry indicates that two plastocyanin molecules bind tightly to the dimeric form of the cyt b6f complex, assuming random insertion of the cytochrome into the bilayer. The structural basis for these results and formation of the proteolipid membrane are discussed.

Cyanobacteria↗
PubMed1998

Protein-protein interaction in electron transfer reactions: the ferredoxin/flavodoxin/ferredoxin:NADP+ reductase system from Anabaena.

Electron transfer reactions involving protein-protein interactions require the formation of a transient complex which brings together the two redox centres exchanging electrons. This is the case for the flavoprotein ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena, an enzyme which interacts with ferredoxin in the photosynthetic pathway to receive the electrons required for NADP+ reduction. The reductase shows a concave cavity in its structure into which small proteins such as ferredoxin can fit. Flavodoxin, an FMN-containing protein that is synthesised in cyanobacteria under iron-deficient conditions, plays the same role as ferredoxin in its interaction with FNR in spite of its different structure, size and redox cofactor. There are a number of negatively charged amino acid residues on the surface of ferredoxin and flavodoxin that play a role in the electron transfer reaction with the reductase. Thus far, in only one case has charge replacement of one of the acidic residues produced an increase in the rate of electron transfer, whereas in several other cases a decrease in the rate is observed. In the most dramatic example, replacement of Glu at position 94 of Anabaena ferredoxin results in virtually the complete loss of ability to transfer electrons. Charge-reversal of positively charged amino acid residues in the reductase also produces strong effects on the rate of electron transfer. Several degrees of impairment have been observed, the most significant involving a positively charged Lys at position 75 which appears to be essential for the stability of the complex between the reductase and ferredoxin. The results presented in this paper provide a clear demonstration of the importance of electrostatic interactions on the stability of the transient complex formed during electron transfer by the proteins presently under study.

Amino Acids↗
PubMed1997

Iron-sulfur cluster cysteine-to-serine mutants of Anabaena -2Fe-2S- ferredoxin exhibit unexpected redox properties and are competent in electron transfer to ferredoxin:NADP+ reductase.

The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.

Anabaena↗
PubMed1997

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants.

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.

Anabaena↗