Intraprotein Electron Transfer Research Articles

Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase

Comparative CO photolysis kinetics studies on wild-type and autoregulatory (AR) insert-deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN–heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase.

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD(*-)) and hydroquinone (FADH(-)) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH(-)*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD(*-)*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.

Ultrafast quenching of tryptophan fluorescence in proteins: Interresidue and intrahelical electron transfer

Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl- and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications.

Free energies for biological electron transfer from QM/MM calculation: method, application and critical assessment

Computer simulations of biological electron transfer reactions are reviewed with a focus on the calculation of reaction free energy (driving force) and reorganization free energy. Then a mixed quantum mechanical/molecular mechanical (QM/MM) approach is described which is designed for computation of these quantities for pure electron transfer reactions with large donor-acceptor separation distances. The method is applied to intra-protein electron transfer in Ru(bpy)(2)(im)His33 cytochrome c and the results compared to experimental data. Several modeling aspects which are important for successful calculation of free energies with QM/MM are discussed in detail.

Formation and Function of Flavin Anion Radical in Cryptochrome 1 Blue-Light Photoreceptor of Monarch Butterfly

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.

Femtochemistry in enzyme catalysis: DNA photolyase

Photolyase uses light energy to split UV-induced cyclobutane pyrimidine dimers in damaged DNA. This photoenzyme encompasses a series of elementary dynamical processes during repair function from early photoinitiation by a photoantenna molecule to enhance repair efficiency, to in vitro photoreduction through aromatic residues to reconvert the cofactor to the active form, and to final photorepair to fix damaged DNA. The corresponding series of dynamics include resonance energy transfer, intraprotein electron transfer, and intermolecular electron transfer, bond breaking-making rearrangements and back electron return, respectively. We review here our recent direct studies of these dynamical processes in real time, which showed that all these elementary reactions in the enzyme occur within subnanosecond timescale. Active-site solvation was observed to play a critical role in the continuous modulation of catalytic reactions. As a model system for enzyme catalysis, we isolated the enzyme-substrate complex in the transition-state region and mapped out the entire evolution of unmasked catalytic reactions of DNA repair. These observed synergistic motions in the active site reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.

Direct Measurement by Laser Flash Photolysis of Intraprotein Electron Transfer in a Rat Neuronal Nitric Oxide Synthase

Intraprotein interdomain electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies have provided a direct determination of the kinetics of IET between the FMN and heme domains in truncated oxyFMN constructs of rat neuronal NOS (nNOS) and murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng, C. J.; Tollin, G.; Holliday, M. A.; Thomas, C.; Salerno, J. C.; Enemark, J. H.; Ghosh, D. K. Biochemistry 2006, 45, 6354-6362. Feng, C. J.; Thomas, C.; Holliday, M. A.; Tollin, G.; Salerno, J. C.; Ghosh, D. K.; Enemark, J. H. J. Am. Chem. Soc. 2006, 128, 3808-3811]. Here, we report the kinetics of IET between the FMN and heme domains in a rat nNOS holoenzyme in the presence and absence of added CaM using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct. The IET rate constant in the presence of CaM is 36 s-1, whereas no IET was observed in the absence of CaM. The kinetics reported here are about an order of magnitude slower than the kinetics in a rat nNOS oxyFMN construct with added CaM (262 s-1). We attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex. This work provides the first direct measurement of CaM-controlled electron transfer between catalytically significant redox couples of FMN and heme in a nNOS holoenzyme.

Conformational and Thermodynamic Control of Electron Transfer in Neuronal Nitric Oxide Synthase

Multiple solution-state techniques have been employed in investigating the nature and control of electron transfer in the context of the proposed "domain shuffle hypothesis" for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. NADPH analogues and fragments have been used to map those regions of this substrate that are important in eliciting a conformational change, observed in both the fluorescence emission of the flavin cofactors of the enzyme and the EPR spectra of the FMN flavosemiquinone state. EPR and UV-visible potentiometric methods have demonstrated a substantial calmodulin-dependent perturbation in the midpoint reduction potentials of the redox couples of both flavin cofactors, in contrast to a previous report [Noble, M. A., et al. (1999) Biochemistry 38, 16413-16418]. These studies support a model in which FMN domain mobility, triggered by Ca2+-calmodulin binding and antagonized by substrate binding, facilitates electron transfer in nitric oxide synthase through conformational change and effects a major change in the midpoint reduction potentials of the flavin redox couples. These results are discussed in light of the recent crystal structure of the NADPH-locked reductase domain.

Redox chemistry of tungsten and iron–sulfur prosthetic groups in Pyrococcus furiosus formaldehyde ferredoxin oxidoreductase

Formaldehyde oxidoreductase (FOR) is one of the tungstopterin iron-sulfur enzymes of the five-membered family of aldehyde oxidoreductases in the hyperthermophilic archaeon Pyrococcus furiosus. In dye-mediated equilibrium redox titrations, the tungsten in active P. furiosus FOR is a two-electron acceptor, W(VI/IV). The intermediate, paramagnetic W(V) state can be trapped only by reduction with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe-4S](2+;+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Electron paramagnetic resonance (EPR) spectroscopy reveals a single "low-potential" W(V) spectrum with gxyz values 1.847, 1.898, and 1.972, and a [4Fe-4S]+ cubane in a spin mixture of S = 1/2 (10%) and S = 3/2 (90%) of intermediate rhombicity (E/D = 0.21, greal = 1.91). The development of this intermediate in vitro is slow even at elevated temperature and with a nominal 50:1 excess of substrate over enzyme presumably owing to the very unfavorable hydration equilibrium of the formaldehyde/methylene glycol couple with KD approximately 10(3). Rapid intermediate formation of enzyme at concentrations suitable for EPR spectroscopy (200 microM) is only obtained with extremely high nominal substrate concentration (1 M formaldehyde) and is followed by a slower phase of denaturation. The premise that the free formaldehyde, and not the methylene glycol, is the enzyme's substrate implies that KM for formaldehyde is 3 orders of magnitude less that the previously reported value.

Comparison of Intra- vs Intermolecular Long-Range Electron Transfer in Crystals of Ruthenium-Modified Azurin

Selective metal-ion incorporation and ligand substitution are employed to control whether electrons tunnel over intra- or intermolecular separations in crystals of P. aeruginosa azurin modified with Ru-polypyridine complexes. Cu(1+)-to-Ru3+ electron transfer (ET) across a specific protein-protein interface in the crystal lattice has a time constant 5-10 times longer than ET between the same donor and acceptor within a single protein (tauET = 5 vs 0.5-1.0 micros). Slower intermolecular ET agrees well with a longer distance between redox centers across the inter-protein (18.9 A) compared to the intra-protein separation (17.0 A) and indicates that the closest donor/acceptor pair dominates crystal ET. Lowering the crystal pH accelerates inter-protein ET (tauET = 1.0 micros) but not intra-protein ET. Faster inter-protein ET likely results from a pH-induced peptide bond flip that perturbs hydrogen bonding in the path between Ru and Cu centers on adjacent molecules.

Intraprotein Electron Transfer in a Two-Domain Construct of Neuronal Nitric Oxide Synthase: The Output State in Nitric Oxide Formation

Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s(-)(1)) was increased approximately 10-fold compared to that in the absence of CaM (22 s(-)(1)). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.

Midpoint Potentials of Hemesaanda3in the Quinol Oxidase fromAcidianus ambivalensare Inverted

The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 +/- 20 mV for the former and 390 +/- 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CuB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO.

Stabilization of Non-productive Conformations Underpins Rapid Electron Transfer to Electron-transferring Flavoprotein

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.

Activationless electron transfer through the hydrophobic core of cytochrome c oxidase

Electron transfer (ET) within proteins occurs by means of chains of redox intermediates that favor directional and efficient electron delivery to an acceptor. Individual ET steps are energetically characterized by the electronic coupling V, driving force DeltaG, and reorganization energy lambda. lambda reflects the nuclear rearrangement of the redox partners and their environment associated with the reactions; lambda approximately 700-1,100 meV (1 eV = 1.602 x 10(-19) J) has been considered as a typical value for intraprotein ET. In nonphotosynthetic systems, functional ET is difficult to assess directly. However, using femtosecond flash photolysis of the CO-poised membrane protein cytochrome c oxidase, the intrinsic rate constant of the low-DeltaG electron injection from heme a into the heme a(3)-Cu(B) active site was recently established at (1.4 ns)(-1). Here, we determine the temperature dependence of both the rate constant and DeltaG of this reaction and establish that this reaction is activationless. Using a quantum mechanical form of nonadiabatic ET theory and common assumptions for the coupled vibrational modes, we deduce that lambda is <200 meV. It is demonstrated that the previously accepted value of 760 meV actually originates from the temperature dependence of Cu(B)-CO bond breaking. We discuss that low-DeltaG, low-lambda reactions are common for efficiently channeling electrons through chains that are buried inside membrane proteins.

Light-induced Electron Transfer in Arabidopsis Cryptochrome-1 Correlates with in Vivo Function

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.

Analysis of the Role of Intraprotein Electron Transfer in Photoreactivation by DNA Photolyase in Vivo

Escherichia coli DNA photolyase contains FADH(-) as the catalytic cofactor. The cofactor becomes oxidized to the FADH(*) blue neutral radical during purification. The E-FADH(*) form of the enzyme is catalytically inert but can be converted to the active E-FADH(-) form by a photoreduction reaction that involves intraprotein electron transfer from Trp306. It is thought that the E-FADH(*) form is also transiently generated during pyrimidine dimer repair by photoinduced electron transfer, and it has been suggested that the FADH(*) that is generated after each round of catalysis must be photoreduced before the enzyme can engage in subsequent rounds of repair. In this study, we introduced the Trp306Phe mutation into the chromosomal gene and tested the non-photoreducible W306F mutant for photorepair in vivo. We find that both wild-type and W306F mutant photolyases carry out at least 25 rounds of photorepair at the same rate. We conclude that photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions.

Electron transfer between hemes in mammalian cytochrome c oxidase

Fast intraprotein electron transfer reactions associated with enzymatic catalysis are often difficult to synchronize and therefore to monitor directly in non-light-driven systems. However, in the mitochondrial respiratory enzyme cytochrome oxidase aa(3), the kinetics of the final electron transfer step into the active site can be determined: reverse electron flow between the close-lying and chemically identical hemes a(3) and a can be initiated by flash photolysis of CO from reduced heme a(3) under conditions where heme a is initially oxidized. To follow this reaction, we used transient absorption spectroscopy, with femtosecond time resolution and a time window extending to 4 ns. Comparison of the picosecond heme a(3)-CO photodissociation spectra under different redox states of heme a shows significant spectral interaction between both hemes, a phenomenon complicating the interpretation of spectral studies with low time resolution. Most importantly, we show that the intrinsic electron equilibration, corresponding to a DeltaG(0) of 45-55 meV, occurs in 1.2 +/- 0.1 ns. This is 3 orders of magnitude faster than the previously established equilibration phase of approximately 3 mus, which we suggest to reflect a change in redox equilibrium closely following CO migration out of the active site. Our results allow testing a number of conflicting predictions regarding this reaction between both experimental and theoretical studies. We discuss the potential physiological relevance of fast equilibration associated with this low-driving-force redox reaction.

Nanobionics of Pharmacologically Active Derivatives of Fullerene C60

The physical–chemical mechanisms of pharmacologic functioning of amino acid derivatives of fullerene C60 (ADF) have been studied. ADF were shown to penetrate through the lipid bilayer of liposomes without destruction of membrane integrity. ADF are able to carry bivalent metal ions through phospholipid bilayer owing to the formation of complexes. It was shown that stereoisomers of ADF selectively penetrate into phospholipid membranes. In contrast to D-isomers, L-isomers penetrate through the phosphatidylcholine membrane into liposome interior. Stereo-specific effect of ADF enantiomers was also observed in reaction of peroxidation of lipids. Besides that, ADF bring about a substantial increase in the catalytic activity of monoaminoxidases A and B. The directed intraprotein electron transfer was studied by creating a donor–acceptor pair in a water solution in the presence of ADF. To realize the intraprotein electron transfer, the model system was produced on the base of apomyoglobin by incorporating ADF (electron acceptor) into the heme pocket of protein instead of removed heme. It was established that the fullerene C60 and its derivatives did not produce specific anti-C60 antibodies, both IgG and IgE classes, while ADF themselves are efficient adjuvants, i.e. they increased the antibody response to poor antigens. Some ADF were found to inhibit the human immunodeficiency virus and human cytomegalovirus infection.

Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans.

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.

Light-induced electron transfer in a cryptochrome blue-light photoreceptor.

Cryptochromes are flavoproteins implicated in multiple blue light-dependent signaling pathways regulating, for example, photomorphogenesis in plants or circadian clocks in animals. Using transient absorption spectroscopy, it is demonstrated that the primary light reactions in isolated Arabidopsis thaliana cryptochrome-1 involve intraprotein electron transfer from tryptophan and tyrosine residues to the excited flavin adenine dinucleotide cofactor.