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     

CW-EPR and ENDOR study of cytochrome c6 from Anabaena PCC 7119

The detailed analysis of the continuous-wave electron paramagnetic resonance and electron nuclear double resonance measurements on cytochrome c(6) from Anabaena PCC7119 reveals several electronic and structural properties of this hemeprotein. The oxidized protein shows two forms that differ in the arrangement of the residues that act as heme axial ligands. Information about the orientation of these residues is obtained for one of the forms, which turns out to differ from that found in the reduced protein from x-ray experiments. The biological significance of these results is discussed.     

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     

Flavodoxin from the nitrogen-fixing cyanobacterium Anabaena PCC 7119

Flavodoxin has been isolated and purified from cultures of the cyanobacterium Anabaena cultivated in a low-iron medium. This flavoprotein has a molecular weight of 20,000 and contains 1 molecule of flavin mononucleotide per mol of protein. Various biochemical characteristics are reported including amino-acid composition, isoelectric point and the fluorescence properties of the apoprotein. The extinction coefficients and isosbestic points were determined for the oxidized and semiquinone forms of flavodoxin. The electron paramagnetic resonance spectrum of the semiquinone exhibited a spectral linewidth of 23 G, which is typical for a neutral flavoprotein semiquinone. Kinetic measurements give a rate constant of 9.6×10⁷ (M^-1 min^-1) for the reduction of flavodoxin in the photosynthetic electron-transport chain by the photosystem I and 6.6×10⁶ for the reaction in which flavodoxin is reduced by ferredoxin-NADP⁺ oxidoreductase. The Michaelis constant for electron donation to nitrogenase by reduced flavodoxin is 8.5 M.Abbreviations FMN flavin mononucleotide - FNR ferredoxin-NADP⁺ oxidoreductase - PSI photosystem I     

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     

Structure of the three-haem core of cytochrome c 551.5 determined by 1H NMR

 The trihaem cytochrome c _551.5, formerly known as cytochrome c ₇, from the organism Desulfuromonas acetoxidans, has been studied in the reduced state by 2D proton NMR. The haem proton resonances were assigned, and several nuclear Overhauser enhancements (NOEs) between resonances arising from different haems were detected and assigned. The relative orientations of the three haems were calculated by fitting both the intensities of the interhaem NOEs and the magnitudes of the ring current shifts of the haem resonances, following the strategy previously used by the authors to reassess the X-ray structure of the haem core in tetrahaem cytochrome c ₃ from Desulfumicrobium baculatum. It is concluded that, although the comparison of the protein sequence with those of the tetrahaem cytochromes c ₃ shows that in cytochrome c _551.5 about 40% of the sequence is deleted, including the region involved in the attachment of the second of the four haems, this does not induce any significant rearrangement of the remaining three haems other than a slight decrease in the iron-iron distance between two of the haems, namely those corresponding to haems I and IV of cytochrome c ₃. Received: 2 February 1996 / Accepted: 26 March 1996     

Unexpected changes in photosystem I function in a cytochrome c6-deficient mutant of the cyanobacterium Synechocystis PCC 6803

Cytochrome c6, the product of the petJ gene, is a photosynthetic electron carrier in cyanobacteria, which transfers electrons to photosystem I and which is synthesised under conditions of copper deficiency to functionally replace plastocyanin. The photosystem I photochemical activity (energy storage, photoinduced P700 redox changes) was examined in a petJ-null mutant of Synechocystis PCC 6803. Surprisingly, photosystem I activity in the petJ-null mutant grown in the absence of copper was not much affected. However, in a medium with a low inorganic carbon concentration and with NH4+ ion as nitrogen source, the mutant displayed growth inhibition. Analysis showed that, especially in the latter, the isiAB operon, encoding flavodoxin and CP43', an additional chlorophyll a antenna, was strongly expressed in the mutant. These proteins are involved in photosystem I function and organisation and are proposed to assist in prevention of overoxidation of photosystem I at its lumenal side and overreduction at its stromal side.     

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     

Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter

A gene coding for a Fur (ferric uptake regulation) protein from the cyanobacterium Anabaena PCC 7119 has been cloned and overexpressed in Escherichia coli. DNA sequence analysis confirmed the presence of a 151-amino-acid open reading frame that showed homology with the Fur proteins reported for the unicellular cyanobacteria Synechococcus 7942 and Synechocystis PCC 6803. Two putative Fur-binding sites were detected in the promoter regions of the fur gene from Anabaena. Partially purified recombinant Fur binds to the flavodoxin promoter as well as its own promoter. This suggests that the Fur gene is autoregulated in Anabaena.     

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.     

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     

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     

Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.     

Redox-Bohr effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms

 Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c ₃ from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e^– step, ideally suited for the coupling role of cytochrome c ₃ to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H₂ as energy source, cytochrome c ₃ can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.     

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     

Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A heterocyst is a terminally differentiated cell of cyanobacteria which is specialized in dinitrogen fixation. Heterocyst differentiation in Anabaena sp. strain PCC 7120 is triggered by deprivation of combined nitrogen in the medium. Although various genes that are upregulated during heterocyst differentiation have been reported, most studies to date were limited to individual or a small number of genes. We prepared microarrays in collaboration with other members of the Anabaena Genome Project. Here we report on the genome-wide expression analysis of the responses to nitrogen deprivation in Anabaena. Many unidentified genes, as well as previously known genes, were found to be upregulated by nitrogen deprivation at various time points. Three main profiles of gene expression were found: genes expressed transiently at an early stage (1-3 hr) of nitrogen deprivation, genes expressed transiently at a later stage (8 hr), and genes expressed when heterocysts are formed (24 hr). We also noted that many of the upregulated genes were physically clustered to form 'expressed islands' on the chromosome. Namely, large, continuous genomic regions containing many genes were upregulated in a coordinated manner. This suggests a mechanism of global regulation of gene expression that involves chromosomal structure, which is reminiscent of eukaryotic chromatin remodelling. The possible implications of this global regulation are discussed.