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.     

Conformational reorganisation in interfacial protein electron transfer

Protein-protein electron transfer (ET) plays an essential role in all redox chains. Earlier studies which used cross-linking and increased solution viscosity indicated that the rate of many ET reactions is limited (i.e., gated) by conformational reorientations at the surface interface. These results are later supported by structural studies using NMR and molecular modelling. New insights into conformational gating have also come from electrochemical experiments in which proteins are noncovalently adsorbed on the electrode surface. These systems have the advantage that it is relatively easy to vary systematically the driving force and electronic coupling. In this review we summarize the current knowledge obtained from these electrochemical experiments and compare it with some of the results obtained for protein-protein ET.     

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.     

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.     

Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites

Benzene, toluene, and o-xylene (BTX) degradation by immobilized Pseudomonas putida F1 of postconsumer agave-fiber/polymer foamed-composites (AFPFC) and suspended cultures was studied under controlled conditions. Analyses using FTIR-ATR and SEM showed that P. putida F1 adhered onto the composite surface and developed a biofilm. In this sense, the AFPFC were successfully used as a support for bacterial immobilization. Both systems, immobilized and suspended cells of P. putida F1, were able to completely degrade benzene and toluene from initial concentrations of 15, 30, 60, and 90 mg l⁻¹. An inhibitory effect of the intermediary catechol from benzene degradation was observed in suspended cultures but it was not presented in the immobilized system. The degradation of o-xylene was partially accomplished in both systems. The Monod equation was used to model the experimental data obtained from the biodegradation kinetics, and they were adequately described with this model.     

Mediated catalysis of <Emphasis Type="Italic">Paracoccus pantotrophus</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase by <Emphasis Type="Italic">P. pantotrophus</Emphasis> pseudoazurin: kinetics of intermolecular electron transfer

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E ⁰′, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 × 10⁵ M⁻¹ s⁻¹ was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.     

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K _m and V _max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.     

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K _m (for UDP-glucose) 0.5–1.0 mM; there was also a minor activity with an unusually high K _m of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K _m values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E_L, E_M and E_H respectively). E_M was the single major contributor to extractable UDPGDH activity when assayed at 0.6–9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E_L-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E_H activity is not due to ADH. At 30 μM UDP-glucose, 20–150 μM UDP-xylose inhibited UDPGDH activity, whereas 5–15 μM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.     

A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase

Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate that the accessibility and electrostatic charge of the active site do not differ in a significant way among the three proteins, but human cytochrome c exhibits some intriguing differences when interacting with cytochrome c oxidase that could be related to the amino acid changes underwent by the latter along evolution.     

Evolution of structure and function of Class I peroxidases

The phylogenetics of Class I of the heme peroxidase-catalase superfamily currently representing over 940 known sequences in all available genomes of prokaryotes and eukaryotes has been analysed. The robust reconstructed tree for 193 Class I peroxidases with 6 selected Class II representatives reveals all main trends of molecular evolution. It suggests how the ancestral peroxidase gene might have been transferred from prokaryotic into eukaryotic genomes. Besides well known families of catalase-peroxidases, cytochrome c peroxidases and ascorbate peroxidases, the phylogenetic analysis shows for the first time the presence of two new well separated clades of hybrid-type peroxidases that might represent evolutionary bridges between catalase-peroxidases and cytochrome c peroxidases (type A) as well as between ascorbate peroxidases and Class II peroxidases (type B). Established structure-function relationships are summarized. Presented data give useful hints on the origin and evolution of catalytic promiscuity and specificity and will be a valuable basis for future functional analysis of Class I enzymes as well as for de novo design.     

Protective action of l-carnitine on cardiac mitochondrial function and structure against fatty acid stress

Cardiovascular risks are frequently accompanied by high serum fatty acid levels. Although recent studies have shown that fatty acids affect mitochondrial function and induce cell apoptosis, l-carnitine is essential for the uptake of fatty acids by mitochondria, and may attenuate the mitochondrial dysfunction and apoptosis of cardiocytes. This study aimed to elucidate the activity of l-carnitine in the prevention on fatty acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoyl-CoA-induced mitochondrial respiration that was observed with l-carnitine was inhibited with oligomycin. The palmitoyl-CoA-induced mitochondrial membrane depolarization and swelling were greatly inhibited by the presence of l-carnitine. In ultrastructural observations, terminally swollen and ruptured mitochondria with little or no distinguishable cristae structures were induced by treatment with palmitoyl-CoA. However, the severe morphological damage in cardiac mitochondria was dramatically inhibited by pretreatment with l-carnitine. Treatment with l-carnitine also attenuated 4-hydroxy-l-phenylglycine- and rotenone-induced mitochondrial swelling even when the l-carnitine could not protect against the decrease in oxygen consumption associated with these inhibitors. Furthermore, l-carnitine completely inhibited palmitoyl-CoA-induced cytochrome c release. We concluded that l-carnitine is essential for cardiac mitochondria to attenuate the membrane permeability transition, and to maintain the ultrastructure and membrane stabilization, in the presence of high fatty acid β-oxidation. Consequently, the cells may be protected against apoptosis by l-carnitine through inhibition of the fatty acid-induced cytochrome c release.     

Isolation and preliminary characterization of new cytochrome c from autotrophic haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

New small cytochrome c (TniCYT) was purified from haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens. The protein was analyzed by mass spectrometry as well as using visible, CD and EPR spectroscopy. It was found that TniCYT is a monomer with a molecular mass of 9461 Da which contains two hemes per molecule. The data of CD and EPR spectroscopy showed that two hemes possess different optical activity and are in distinct, high and low spin states. TniCYT was also demonstrated to have unusual characteristics in the visible spectrum, namely, the splitting of characteristic peaks was observed in α- and β-bands of the heme spectrum when the reduced form of cytochrome was analyzed. The α-band has two peaks with maximum at 548 and 556 nm whereas the β-band showes ones at 520 and 528 nm. According to the MALDI finger-print analysis, the new cytochrome has a unique amino acid sequence.     

Electrochemistry of cytochrome c immobilized on cardiolipin-modified electrodes: A probe for protein–lipid interactions

Electrochemistry of cytochrome c (cyt c) immobilized on a cardiolipin (CL)/phosphatidylcholine (PC) film supported on a glassy carbon electrode was investigated using variable-frequency AC voltammetry. At low ionic strength, we observed two redox-active subpopulations characterized by distinct values of potential (E_1/2) and electron transfer rate constant (k_ET). At high ionic strength, only one subpopulation was detected, consistent with the existence of very stable cyt c–CL adducts, most probably formed by hydrophobic interactions between the protein and the fatty acid (FA) chains carried by CL. This subpopulation exhibits a comparatively high k_ET value (> 300 s^− 1) apparently changing with the structure of the FA chains of CL, i.e. 18:2(n − 6) or 14:0. Our study suggests that electrochemistry can be a useful technique for probing protein–lipid interactions, and more particularly the role played by the specific structure of the FA chains of CL on cyt c binding.     

The heme domain of cellobiose oxidoreductase: a one-electron reducing system

Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases.     

Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent.     

Dynamics of mitochondrial structure during apoptosis and the enigma of Opa1

“The large scale remodeling of mitochondria during apoptosis is a necessary step for the complete release of cytochrome c” has been a tenet since 2002. However, more recent findings strongly indicate that the large-scale remodeling previously described actually takes place after the release of cytochrome c and in a caspase-dependent manner, bringing into question whether mitochondria remodeling is necessary. In a more recent article, however, it was shown that a much more subtle form of remodeling is taking place which is only observable by electron tomography. In the Bcl-2 inhibitable Bax/Bak-dependent intrinsic pathway of apoptosis, the release of cytochrome c from mitochondria is a consequence of two carefully coordinated events: formation of outer membrane pores and opening of crista junctions triggered by Opa1 oligomer disassembly, and both steps are necessary for the complete release of cytochrome c. We review the recent literature pertaining to the coordinated release of cytochrome c during cell death.     

Temperature and metabolism in Leishmania. 3. Some dehydrogenases of L. donovani, L. mexicana, and L. tarentolae

The effects of temperature on four dehydrogenases in homogenates of promastigotes of Leishmania donovani (several strains), L. mexicana, and L. tarentolae were studied.     

D-3-hydroxybutyrate dehydrogenase from Pseudomonas fragi: molecular cloning of the enzyme gene and crystal structure of the enzyme

The gene coding for d-3-hydroxybutyrate dehydrogenase (HBDH) was cloned from Pseudomonas fragi. The nucleotide sequence contained a 780 bp open reading frame encoding a 260 amino acid residue protein. The recombinant enzyme was efficiently expressed in Escherichia coli cells harboring pHBDH11 and was purified to homogeneity as judged by SDS-PAGE. The enzyme showed a strict stereospecificity to the D-enantiomer (3R-configuration) of 3-hydroxybutyrate as a substrate. Crystals of the ligand-free HBDH and of the enzyme-NAD+ complex were obtained using the hanging-drop, vapor-diffusion method. The crystal structure of the HBDH was solved by the multiwavelength anomalous diffraction method using the SeMet-substituted enzyme and was refined to 2.0 A resolution. The overall structure of P.fragi HBDH, including the catalytic tetrad of Asn114, Ser142, Tyr155, and Lys159, shows obvious relationships with other members of the short-chain dehydrogenase/reductase (SDR) family. A cacodylate anion was observed in both the ligand-free enzyme and the enzyme-NAD+ complex, and was located near the catalytic tetrad. It was shown that the cacodylate inhibited the NAD+-dependent D-3-hydroxybutyrate dehydrogenation competitively, with a Ki value of 5.6 mM. From the interactions between cacodylate and the enzyme, it is predicted that substrate specificity is achieved through the recognition of the 3-methyl and carboxyl groups of the substrate.     

Reproductive function for a C-terminus extended, male-transmitted cytochrome c oxidase subunit II protein expressed in both spermatozoa and eggs

Our previous study documented expression of a male-transmitted cytochrome c oxidase subunit II protein (MCOX2), with a C-terminus extension (MCOX2e), in unionoidean bivalve testes and sperm mitochondria. Here, we present evidence demonstrating that MCOX2 is seasonally expressed in testis, with a peak shortly before fertilization that is independent of sperm density. MCOX2 is localized to the inner and outer sperm mitochondrial membranes and the MCOX2 antibody's epitope is conserved across >65 million years of evolution. We also demonstrate the presence of male-transmitted mtDNA and season-specific MCOX2 spatial variation in ovaries. We hypothesize that MCOX2 plays a role in reproduction through gamete maturation, fertilization and/or embryogenesis.