Factors influencing the energetics of electron and proton transfers in proteins. What can be learned from calculations

A protein structure should provide the information needed to understand its observed properties. Significant progress has been made in developing accurate calculations of acid/base and oxidation/reduction reactions in proteins. Current methods and their strengths and weaknesses are discussed. The distribution and calculated ionization states in a survey of proteins is described, showing that a significant minority of acidic and basic residues are buried in the protein and that most of these remain ionized. The electrochemistry of heme and quinones are considered. Proton transfers in bacteriorhodopsin and coupled electron and proton transfers in photosynthetic reaction centers, 5-coordinate heme binding proteins and cytochrome c oxidase are highlighted as systems where calculations have provided insight into the reaction mechanism.     

Effects of heme on the structure of the denatured state and folding kinetics of cytochrome b562

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Stability enhancement of cytochrome c through heme deprotonation and mutations

The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.     

Desulfovibrio desulfuricans G20 tetraheme cytochrome structure at 1.5 Angstrom and cytochrome interaction with metal complexes

The structure of the type I tetraheme cytochrome c(3) from Desulfovibrio desulfuricans G20 was determined to 1.5 Angstrom by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated by circular dichroism studies. Again, only small changes in protein structure were documented. The location of the molybdate ion near heme IV in the crystal structure suggested heme IV as the site of electron exit from the reduced cytochrome and implicated Lys14 and Lys56 in binding. Analysis of structurally conserved water molecules in type I cytochrome c(3) crystal structures identified interactions predicted to be important for protein stability and possibly for intramolecular electron transfer among heme molecules.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.     

Increasing the conformational stability by replacement of heme axial ligand in c-type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M⁻¹ s⁻¹ and 0.34 ± 0.15 s⁻¹, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min⁻¹ at pH 6.0.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b_6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b₆f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c_i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b₆f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

Electron flow through metalloproteins

Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor-acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Characterization of a cytochrome b(558) ferric/cupric reductase from rabbit duodenal brush border membranes

Iron and probably also copper are absorbed by the intestine in their reduced form. A b-type cytochrome, Dcytb, has recently been cloned from mouse and has been proposed to be the corresponding reductase. However, the nature of the cytochrome and the reduction reaction remain unknown. Here we describe the isolation and functional characterization of a novel b-type cytochrome from rabbit enterocytes. The 33 kDa heme protein was solubilized from brush border membranes with Triton X-100 and purified by successive ion exchange chromatography and hydrophobic interaction chromatography. Spectroscopic analysis of the heme revealed a b(558) cytochrome. The purified hemoprotein exhibited ascorbate-stimulated reduction of iron(III) and copper(II). The rate constants, k(1), for these reactions were 1.38 +/- 0.12 and 0.64 +/- 0.16 min(-1), respectively. Cytochrome b(558) may be the rabbit Dcytb homologue. A novel mechanism of how cytochrome b(558) could shuttle electrons from cytoplasmic ascorbate to luminal dehydroascorbate is proposed.     

Heterologous expression and in vitro assembly of the transmembrane cytochrome b6

Folding and assembly studies with alpha-helical membrane proteins are often hampered by the absence of high-level expression systems as well as by missing suitable in vitro refolding procedures. Experimental constraints and requirements for heterologous expression and in vitro assembly of cytochrome b6 have been examined and conditions for in vitro reconstitutions of the protein have been optimized. Cytochrome b6 can serve as an excellent model system for in vitro studies on the dynamic interplay of an apo-protein and heme cofactors during assembly of a transmembrane b-type cytochrome. In vitro assembled cytochrome b6 binds two hemes with different midpoint potentials and both ferri as well as ferro heme bind to the apo-cytochrome. However, the ferro cytochrome appears to be less stable than the ferri form.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

Structure of cytochrome c6A, a novel dithio-cytochrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications for function

Cytochrome c6A is a unique dithio-cytochrome present in land plants and some green algae. Its sequence and occurrence in the thylakoid lumen suggest that it is derived from cytochrome c6, which functions in photosynthetic electron transfer between the cytochrome b6f complex and photosystem I. Its known properties, however, and a strong indication that the disulfide group is not purely structural, indicate that it has a different, unidentified function. To help in the elucidation of this function the crystal structure of cytochrome c6A from Arabidopsis thaliana has been determined in the two redox states of the heme group, at resolutions of 1.2 A (ferric) and 1.4 A (ferrous). These two structures were virtually identical, leading to the functionally important conclusion that the heme and disulfide groups do not communicate by conformational change. They also show, however, that electron transfer between the reduced disulfide and the heme is feasible. We therefore suggest that the role of cytochrome c6A is to use its disulfide group to oxidize dithiol/disulfide groups of other proteins of the thylakoid lumen, followed by internal electron transfer from the dithiol to the heme, and re-oxidation of the heme by another thylakoid oxidant. Consistent with this model, we found a rapid electron transfer between ferro-cytochrome c6A and plastocyanin, with a second-order rate constant, k2=1.2 x 10(7) M(-1) s(-1).