Metal-binding properties of an engineered purple CuA center in azurin

A CUA center engineered into Pseudomonas aeruginosa azurin was studied by metal substitution. Metal-binding properties were determined by electronic absorption (UV-vis) and electrospray ionization mass spectrometry (ESI-MS). The metal-binding site readily binds thiophilic metal ions, such as Hg(II), Ag(I), Cu(I), Cd(II), and Au(I). Harder metal ions, like Co(II), bind to apo-CuA-azurin only under basic conditions (pH 9.1-9.2). The results obtained from these studies indicate that two factors influence metal binding in CuA azurin: (1) the site favors metal combinations which produce an overall +3 charge, and (2) the site binds soft, thiophilic metal ions. The results demonstrate the remarkable ability of the CuA center to maintain valence delocalization of its native metal ions and to ensure redox accessibility of only one of the two redox couples (i.e., [Cu(1.5)...Cu(1.5)]<==> [Cu(I)...Cu(I)]) under physiological conditions. These findings may lead to the preparation of new metal ion derivatives and can serve as a basis for understanding this efficient electron transfer center.     

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.     

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.     

Electronic structure contributions to electron transfer in blue Cu and CuA

The experimentally determined electronic structures of mononuclear blue Cu and binuclear Cu(A) centers are summarized and their relation to intra- and inter-protein electron transfer (ET) kinetics are described. Specific contributions of the electronic structures of these two broad classes of Cu ET proteins to H(AB), lambda, and deltaE degrees are discussed. Also, the role of the protein structure in determining key geometric features which define the electronic structures of the metal sites in these proteins is considered.     

Structural basis of the drastically increased initial electron transfer rate in the reaction center from a Rhodopseudomonas viridis mutant described at 2.00-A resolution

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-A resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the "special pair" bacteriochlorophyll, D(L), is rotated, and its acetyl oxygen is found 1.1 A closer to the bacteriochlorophyll-Mg(2+) of the other special pair bacteriochlorophyll, D(M). The rotation of this acetyl group and the increased interaction between the D(L) ring I acetyl oxygen and the D(M)-Mg(2+) provide the structural basis for the previously observed 80-mV decrease in the D(+)/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, B(A). The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, Q(B).     

NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of approximately 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.     

Structural basis for electron and methyl-group transfer in a methyltransferase system operating in the reductive acetyl-CoA pathway

Several anaerobic acetogenic, methanogenic, hydrogenogenic, and sulfate-reducing microorganisms are able to use the reductive acetyl-CoA (Wood-Ljungdahl) pathway to convert CO₂ into biomass. The reductive acetyl-CoA pathway consists of two branches connected by the Co/Fe-containing corrinoid iron-sulfur protein (CoFeSP), which transfers a methyl group from a methyltransferase (MeTr)/methyltetrahydrofolate (CH₃-H₄ folate) complex to the reduced Ni-Ni-[4Fe-4S] cluster (cluster A) of acetyl-CoA synthase. We investigated the CoFeSP and MeTr couple of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans and show that the two proteins are able to catalyze the methyl-group transfer reaction from CH₃-H₄ folate to the Co(I) center of CoFeSP. We determined the crystal structures of both proteins. The structure of CoFeSP includes the previously unresolved N-terminal domain of the large subunit of CoFeSP, revealing a unique four-helix-bundle-like architecture in which a [4Fe-4S] cluster is shielded by hydrophobic amino acids. It further reveals that the corrinoid and the [4Fe-4S] cluster binding domains are mobile, which is mandatory for the postulated electron transfer between them. Furthermore, we solved the crystal structures of apo-MeTr, CH₃-H₄-folate-bound MeTr, and H₄-folate-bound MeTr, revealing a substrate-induced closure of the CH₃-H₄ folate binding cavity of MeTr. We observed three different conformations of Asn200 depending on the substrate bound in the active site, demonstrating its conformational modulation by hydrogen-bonding interactions with the substrate. The observed flexibility could be essential to stabilize the transition state during methyl-group transfer. The conformational space and role of Asn200 are likely conserved in homologous cobalamin-dependent MeTrs such as methionine synthase.     

Ligand replacement study at the His120 site of purple CuA azurin

The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X = Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn 119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn 119X (X = Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.     

Kinetics of electron transfer between plastocyanin and the soluble CuA domain of cyanobacterial cytochrome c oxidase

It has been shown that efficient functioning of photosynthesis and respiration in the cyanobacterium Synechocystis PCC 6803 requires the presence of either cytochrome c6 or plastocyanin. In order to check whether the blue copper protein plastocyanin can act as electron donor to cytochrome c oxidase, we investigated the intermolecular electron transfer kinetics between plastocyanin and the soluble CuA domain (i.e. the donor binding and electron entry site) of subunit II of the aa3-type cytochrome c oxidase from Synechocystis. Both copper proteins were expressed heterologously in Escherichia coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (5.1+/-0.2) x 10(4) M(-1) s(-1) and (8.5+/-0.4) x 10(5) M(-1) s(-1), respectively (20 mM phosphate buffer, pH 7). This corresponds to an apparent equilibrium constant of 0.06 in the physiological direction (reduction of CuA), which is similar to Keq values calculated for the reaction between c-type cytochromes and the soluble fragments of other CuA domains. The potential physiological role of plastocyanin in cyanobacterial respiration is discussed.     

Rates and Equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse heme a --> CuA process were found to be 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.     

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

To investigate the contribution of tryptophan-121 (Trp121) residue to the structure and function of soluble CuA domain of cytochrome c oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deleted mutant of the soluble domain of Paracoccus versutus cytochrome c oxidase, were constructed and expressed in Escherichia coli BL21 (DE3). Optical spectral studies showed that both the coordination structure of the CuA center and the secondary structure of the protein were changed significantly in the Leu substitution and deletion mutants of Trp121. Their electron transfer activity with cytochrome c was inhibited severely, as shown in stopped-flow kinetic studies. However, the CuA center can be reconstructed in the Trp121Tyr mutant although its stability decreases compared with the wild-type protein. This mutant keeps the same secondary structure as the wild-type protein, but can only transfer electrons with cytochrome c at a rate of one-seventh-fold. Based on the information on the structure, we also investigated and analyzed the possible factors that affect electron transfer. It appears that the aromatic ring, the size of the side chain and the hydrogen bonding ability of the Trp121 are crucial to the structure and function of the soluble CuA domain.     

Comparative and evolutionary aspects of the photosynthetic electron transfer system of purple bacteria

The cyclic electron transfer system in purple bacteria is composed of the photosynthetic reaction center, the cytochromebc ₁ complex, cytochromec ₂, and ubiquinone. These components share many characteristics with those of photosynthesis and respiration in other organisms. Studies of the cyclic electron transfer system have provided useful insights about the evolution and general mechanisms of membranous energy-conserving systems. The photosynthetic system in purple bacteria may represent a prototype of highly efficient, energy-conserving electron transfer systems in the organisms. Recipient of the Botanical Society Award of Young Scientists, 1992     

Structural basis underlying the electron transfer features of a blue copper protein auracyanin from the photosynthetic bacterium Roseiflexus castenholzii

Photosynthesis Research - Auracyanin (Ac) is a blue copper protein that mediates the electron transfer between Alternative Complex III (ACIII) and downstream electron acceptors in both fort chains...     

In vitro and in vivo electron transfer to the triheme cytochrome subunit bound to the photosynthetic reaction center complex in the purple bacterium Rhodovulum sulfidophilum.

The cytochrome subunit bound to the photosynthetic reaction center (RC) complex in Rhodovulum sulfidophilum lacks one heme-binding motif (CXXCH) out of four motifs found in other purple bacteria resulting in the absence of the most distal heme from the RC-core complex (S. Masuda et al., J. Biol. Chem. 274 (1999) 10795). Cytochrome c(2), which acts as the electron donor to the RC was purified, and its gene was cloned and sequenced. The redox midpoint potential of cytochrome c(2) was determined to be E(m)=357 mV. The photo-oxidation and re-reduction of purified cytochrome c(2) were observed in the presence of membrane preparations. Flash-induced photo-oxidation and re-reduction of the RC-bound cytochrome were also observed in intact cells. Despite the unusual nature of the RC-bound cytochrome subunit, the cyclic electron transfer system in Rdv. sulfidophilum was shown to be similar to those in other purple bacteria.     

Electrostatic modulation of electron transfer in the active site of heme peroxidases

 The heme enyzmes cytochrome c peroxidase (CCP) and pea cytosolic ascorbate peroxidase (APX) show a high level of sequence identity. The main difference near the active sites is the presence of a cation binding site in APX located about 1 nm from the Trp-179 side chain, which is hydrogen-bonded to Asp-208. It is possible that this difference in electrostatics provided by the protein environment is an essential determinant of the stabilization of the ion-pair or neutral form of the Trp...Asp couple in APX and CCP. Semiempirical molecular orbital calculations support the hypothesis that the position of the moving proton inside the couple influences the location of the free electron, leading to radical formation either on the heme or on the Trp side chain of these enzymes. Received, accepted: 26 November 1996     

Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase

Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways.     

Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein

Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.     

Structural basis of the lipid transfer mechanism of phospholipid transfer protein (PLTP)

Human phospholipid transfer protein (PLTP) mediates the transfer of phospholipids among atheroprotective high-density lipoproteins (HDL) and atherogenic low-density lipoproteins (LDL) by an unknown mechanism. Delineating this mechanism would represent the first step towards understanding PLTP-mediated lipid transfers, which may be important for treating lipoprotein abnormalities and cardiovascular disease. Here, using various electron microscopy techniques, PLTP is revealed to have a banana-shaped structure similar to cholesteryl ester transfer protein (CETP). We provide evidence that PLTP penetrates into the HDL and LDL surfaces, respectively, and then forms a ternary complex with HDL and LDL. Insights into the interaction of PLTP with lipoproteins at the molecular level provide a basis to understand the PLTP-dependent lipid transfer mechanisms for dyslipidemia treatment.