The solution structure of cytochrome c 6 from the green alga Monoraphidium braunii

 The three-dimensional structure in solution of the reduced form of cytochrome c ₆ from the green alga Monoraphidium braunii has been solved through NMR data. Cytochrome c ₆ acts as a small mono-heme electron carrier protein between the two membrane-embedded complexes cytochrome f and photosystem I. The structure was determined using 1278 relevant interproton NOEs out of 1776 assigned NOEs with distance geometry (DG) calculations which included 36 stereospecific assignments and 20 experimentally found angle constraints. The family of structures obtained from the DG calculations was subjected to energy minimization and molecular dynamics simulation using previously defined force field parameters for the heme and its ligands. In all stages of the calculations, the solution structure is well defined and similar to the now available X-ray structure. Received: 18 January 1996 / Accepted: 19 April 1996     

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

 A soluble monoheme c–type cytochrome c ₆ has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP⁺reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c ₆ cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c ₆ at pH 6.7 was determined to be 338 mV and is pH dependent, with pK _a ^ox=8.4±0.1 and pK _a ^red≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and ¹H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pK _a of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c ₆ is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation. Received: 17 May 1996 / Accepted: 13 January 1997     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

Expression of 15N-labeled eukaryotic cytochrome c in Escherichia coli

 We present a simple and inexpensive method for producing ¹⁵N-labeled Saccharomyces cerevisiae iso-1-cytochrome c in Escherichia coli. The labeled protein gives excellent NMR spectra. Received: 18 December 1998 / Accepted: 27 January 1999     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c 3

 Reduction of the haems in tetrahaem cytochromes c ₃ is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c ₃ form a structural motif that is present in all cytochromes c ₃ and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c ₃. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c ₃. NMR studies of F20I cytochrome c ₃ demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c ₃ backbone, which is also very close to haem I, had no effect on the network of cooperativities. Received: 25 June 1996 / Accepted: 26 August 1996     

Theoretical studies on the redox-Bohr effect in cytochrome c 3 from Desulfovibrio vulgaris Hildenborough

 The pH dependence of the redox potentials in the tetrahemic cytochrome c ₃ from Desulfovibrio vulgaris Hildenborough (redox-Bohr effect) is here investigated using continuum electrostatics methods. The redox-Bohr effect seems to be associated with changes in the protonation state of charged residues in the protein, but the exact residues had not been identified. The global pK _a of this phenomenon is dependent on the redox state of the molecule, and the influence of the pH on the microscopic potential of each heme has been experimentally quantified. The availability of detailed experimental data provides us with important and unique guides to the performance of ab initio pK _a calculations aiming at the identification of the groups involved. These calculations were performed in several redox states along the reduction pathway, with the double objective of finding groups with redox-linked pK _a shifts, and absolute pK _as compatible with the redox-Bohr effect. The group with the largest pK _a shift along the reduction pathway is propionate D from heme I. Its effect on the redox potential of individual hemes, as calculated by electrostatic calculations, correlates very well with the experimental order of influence, making it a likely candidate. Abnormal titration of the same propionate has been experimentally observed on a homologous cytochrome c ₃ from a different strain, thus strengthening the theoretical result. However, its absolute calculated pK _a in the fully oxidised cytochrome is outside the zone where the phenomenon is known to occur, but the calculation shows a strong dependence on small conformational changes, suggesting large uncertainties in the calculated value. A group with a pK _a value within the experimentally observed range is propionate D from heme IV. Its influence on the redox potential of the hemes does not correlate with the experimental order, indicating that, although it may be one of the possible players on the phenomenon, it cannot be solely responsible for it. Mutation of the Lys45 residue is suggested as an indirect way of probing the importance of the propionate D from heme I in the mechanism. Non-heme groups may also be involved in this process; our calculations indicate His67 and the N-terminal as groups that may play a role. Accuracy and applicability of current continuum electrostatic methods are discussed in the context of this system. Received: 27 March 1997 / Accepted: 19 August 1997     

Complete isomer-specific 1H and 13C NMR assignments of the heme resonances of rat liver outer mitochondrial membrane cytochrome b 5

 Singly and doubly labeled δ-aminolevulinic acid derivatives were used to prepare rat liver outer mitochondrial membrane (OM) cytochrome b ₅ containing a ¹³C-labeled heme active site. A variety of NMR experiments, including HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NOESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-α and -β carbons in both isomeric forms of the paramagnetic active center of OM cytochrome b ₅. Because the long interpulse delays in the HMBC experiment have a detrimental effect on the detectability of fast relaxing paramagnetically affected resonances, INADEQUATE is proposed as the experiment of choice for assigning quaternary carbons in paramagnetic hemes with carefully chosen macrocycle labeling patterns. Furthermore, the applicability of the INADEQUATE experiment to paramagnetic heme active sites should be facilitated greatly by the availability of biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes. Received: 21 September 1998 / Accepted: 25 November 1998     

Resonance Raman characterization of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

 Di-heme Pseudomonas stutzeri cytochrome c ₄ has been characterized by electronic absorption and resonance Raman spectroscopies in the ferric and ferrous forms at pH 7.5 and at room temperature. The data indicate that the two hemes are inequivalent. It is proposed that the N-terminal contains a more relaxed heme as a consequence of the relative orientation of the methionine and histidine ligands with respect to the N-Fe-N directions of the heme plane. This causes a weakening of the Fe-S bond with concomitant partial dissociation of the methionine and the formation of an Fe-aquo bond. Heme group relaxation is further accompanied by less distortion of the heme group than that associated with cytochrome c, expansion of the "core" and a negative shift of the redox potential. Received: 17 December 1996 / Accepted: 6 March 1997     

Location of a cytochrome c binding site on the surface of flavocytochrome b 2

Saccharomyces cerevisiae flavocytochrome b ₂ couples the oxidation of L-lactate to the reduction of cytochrome c. The second-order rate constant for cytochrome c reduction by flavocytochrome b ₂ depends on the rate of complex formation and is sensitive to ionic strength. Mutations in the heme domain of flavocytochrome b ₂ (Glu63→Lys, Asp72→Lys and the double mutation Glu63→Lys:Asp72→Lys) have significant effects on the reaction with cytochrome c, implicating these residues in complex formation. This kinetic information has been used to guide molecular modelling studies, which are consistent with there being no one single best-configuration. Rather, there is a set of possible complexes in which the docking-face of cytochrome c can approach flavocytochrome b ₂ in a variety of orientations. Four cytochromes c can be accommodated on the flavocytochrome b ₂ tetramer, with each cytochrome c forming interactions with only one flavocytochrome b ₂ subunit. All the models involve residues 72 and 63 on flavocytochrome b ₂ but in addition predict that Glu237 may also be important for complex formation. These acidic residues interact with the basic residues 13, 27 and 79 on cytochrome c. Through this triangle of interactions runs a possible σ-tunnelling pathway for electron transfer. This pathway starts with the imidazole ring of His66 (a ligand to the heme-iron of flavocytochrome b ₂) and ends with the ring of Pro68, which is in van der Waals contact with the cytochrome c heme. In total, the edge-to-edge "through space" distance from the imidazole ring of His66 to the C3C pyrrole ring of cytochrome c is 13.1?Å.     

The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example

 The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only. Received: 11 July 1995 / Accepted: 30 October 1995     

Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated

 The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H₂O₂ to H₂O with oxidation of cytochrome c ₅₅₀ and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4′-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca²⁺ is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem. Received: 29 April 1998 / Accepted: 3 September 1998     

Solution structure of reduced horse heart cytochrome c

 In the frame of a broad study on the structural differences between the two redox forms of cytochromes to be related to the electron transfer process, the NMR solution structure of horse heart cytochrome c in the reduced form has been determined. The structural data obtained in the present work are compared to those already available in the literature on the same protein and the presence of conformational differences is discussed in the light of the experimental method employed for the structure determination. Redox-state dependent changes are analyzed and in particular they are related to the role of propionate-7 of the heme. Also some hydrogen bonds are changed upon reduction of the heme iron. A substantial similarity is observed for the backbone fold, independently of the oxidation state. At variance, some meaningful differences are observed in the orientation of a few side chains. These changes are related to those found in the case of the highly homologous cytochrome c from Saccharomyces cerevisiae. The exchangeability of the NH protons has been investigated and found to be smaller than in the case of the oxidized protein. We think that this is a characteristic of reduced cytochromes and that mobility is a medium for molecular recognition in vivo. Received: 8 June 1998 / Accepted: 13 October 1998     

Comparative redox and pK a calculations on cytochrome c 3 from several Desulfovibrio species using continuum electrostatic methods

 A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c ₃ family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pK _a calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c ₃ redox potentials in all the c ₃ molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c ₃ family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c ₃ from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely. Received: 31 August 1998 / Accepted: 20 November 1998     

Insights into the alkaline transformation of ferricytochrome c from (1)H NMR studies in 30% acetonitrile-water

Recently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous-nonaqueous media, resulting in the formation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One-dimensional (1D) and two-dimensional (2D) (1)H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline-like forms in 30% acetonitrile-water solution. A native-like (M80-ligated) III* form, two lysine-ligated forms (IVa* and IVb*), and a hydroxide-ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D (1)H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20 degrees C and 35 degrees C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for the hydroxide-ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.     

Redox-Bohr effect in electron/proton energy transduction: cytochrome c 3 coupled to hydrogenase works as a 'proton thruster' in Desulfovibrio vulgaris

 A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c ₃, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement. Received: 2 April 1997 / Accepted: 23 May 1997     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.