Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c.

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.     

Evolutionary change of the heme c electronic structure: ferricytochrome c-551 from Pseudomonas aeruginosa and horse heart ferricytochrome c.

Individual assignments of the ¹H n.m.r. lines of heme c in reduced and oxidized cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ were obtained by nuclear Overhauser enhancement and saturation transfer experiments. Comparison with the corresponding data on horse heart cytochrome c showed that the locations of high spin density on the heme c periphery as well as the in-plane principal axes x and y of the electronic g-tensor are rotated by approximately 90° in ferricytochrome c-551 relative to horse ferricytochrome c. High spin density in ferricytochrome c-551 is thus localized on the pyrrole ring III. While this pyrrole ring is well shielded in the interior of mammalian-type cytochromes c, it is more easily accessible in cytochrome c-551. It is suggested that this evolutionary change of the heme c electronic structure would be compatible with the hypothesis that the electron transfer in both species is via solvent exposed peripheral ring carbon atoms.     

Assignment of 1H and 13C hyperfine-shifted resonances for tuna ferricytochrome c.

Tuna ferricytochrome c has been used to demonstrate the potential for completely assigning 1H and 13C strongly hyperfine-shifted resonances in metalloprotein paramagnetic centers. This was done by implementation of standard two-dimensional NMR experiments adapted to take advantage of the enhanced relaxation rates of strongly hyperfine-shifted nuclei. The results show that complete proton assignments of the heme and axial ligands can be achieved, and that assignments of several strongly shifted protons from amino acids located close to the heme can also be made. Virtually all proton-bearing heme 13C resonances have been located, and additional 13C resonances from heme vicinity amino acids are also identified. These results represent an improvement over previous proton resonance assignment efforts that were predicated on the knowledge of specific assignments in the diamagnetic protein and relied on magnetization transfer experiments in heterogeneous solutions composed of mixtures of diamagnetic ferrocytochrome c and paramagnetic ferricytochrome c. Even with that more complicated procedure, complete heme proton assignments for ferricytochrome c have never been demonstrated by a single laboratory. The results presented here were achieved using a more generally applicable strategy with a solution of the uniformly oxidized protein, thereby eliminating the requirement of fast electron self-exchange, which is a condition that is frequently not met.     

Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome.

Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed.     

Preliminary 1H-NMR studies of the interaction between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway

The complex formation of two electron transfer proteins, cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway, has been shown by 1H-NMR spectroscopy. Presence of ferredoxin I produces ferricytochrome c3 1H-NMR spectrum modifications. The chemical shift of perturbated heme methyl resonances has been used to determine the stoichiometry of the complex. At pH 7.6 and 20 degrees C, the two proteins were found to form a complex 1:1 with an association constant, KA, of 10(4) M-1. Two of the four hemes are affected by presence of ferredoxin I and may be involved in the electron transfer sites. The heme methyl resonances are average resonances of free and bound cytochrome c3 resonances, indicating a fast exchange process on the NMR time scale.     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aliphatic amino acids

The aliphatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. There are eight firmly assigned methyl resonances of ferrocytochrome c and seven firmly assigned methyl resonances of ferricytochrome c.     

13C and proton NMR studies of horse cytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of the 55 methyl groups of horse cytochrome c have been determined over a range of temperatures both in the diamagnetic ferrocytochrome and in the paramagnetic ferricytochrome. Specific assignments of many proton resonances have been published previously and all of the remaining methyl proton resonances are now specifically assigned. The corresponding 13C assignments follow directly, including those of contact shifted 13C resonances which are reported for the first time.     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

L-Lactate cytochrome c reductase: rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly

This study is part of a series aimed at the characterization of individual steps of electron transfer taking place between prosthetic flavin, heme b2, heme c within active sites and complexes. After rapid mixing of ferricytochrome c with partially reduced flavocytochrome b2, the reaction is followed at the level of two reactants, cytochrome b2 and cytochrome c. In order to define the proper reactivity of flavosemiquinone, conditions under which this form is highly stabilized (presence of pyruvate) have been chosen. With the help of simulations, it has been possible to characterize a rapid step of electron transfer from cytochrome b2 to cytochrome c within a complex (at approx. 70% saturation) and a slow step k = 5 s-1 assigned to cytochrome b2 reduction by flavosemiquinone within the active site of the pyruvate-liganded enzyme.     

Proton NMR comparison of the Saccharomyces cerevisiae ferricytochrome c isozyme-1 monomer and covalent disulfide dimer

Proton NMR studies of Saccharomyces cerevisiae (bakers yeast) isozyme-1 monomer and dimer ferricytochrome c have been carried out. The dimer is formed via a disulfide bridge between the Cys-102 residues of monomer proteins. Nuclear Overhauser effect (NOE) experiments have led to resonance assignments for many of the heme and axial ligand (Met-80; His-18) protons in both protein forms. Resonances of the following amino acids have also been assigned in both forms: Phe-10; Pro-30; Phe-82; Trp-59; Leu-68. The proton NOE connectivity patterns of the monomer of yeast isozyme-1 ferricytochrome c are similar to those of horse, tuna, and yeast isozyme-2 ferricytochromes c, even though the observed hyperfine resonance spectra are significantly different for the various cytochromes. The pattern of dimer proton hyperfine resonances is distinct from the isozyme-1 monomer pattern, which indicates that the formation of a disulfide bridge via Cys-102 is detected at the heme site, approximately 10 A distant. It appears that a specific structural change is induced upon dimerization, which, in turn, causes specific perturbations in the vicinity of the heme. However, the general features of the NOE connectivity pattern in the dimer are the same as for the monomer indicating that dimerization does not result in drastic structural disruption. Furthermore, the 1H NMR spectrum of the dimer can be mimicked by the monomer form that results when the -SH group of Cys-102 is chemically modified with certain types of bulky, or hydrophilic reagents (i.e. 5,5'-dithiobis[2-nitrobenzoate], indicating that perturbations of the yeast isozyme-1 ferricytochrome c proton resonance spectrum observed upon dimerization are essentially due to changes in intramolecular, rather than intermolecular, interactions. These results suggest that a possible regulatory site for yeast isozyme-1 cytochrome c exists at position 102, which could conceivably have a physiological role in altering the conformation of the molecule.     

Cytochrome c reductase purified from Crithidia fasciculata contains an atypical cytochrome c1.

Cytochrome c reductase purified from the trypanosomatid Crithidia fasciculata retained antimycin A sensitivity and catalyzed the reduction of horse heart ferricytochrome c in the presence of reduced coenzyme Q10. The complex contained heme b and heme c1 in a ratio of 2:1. Nine major protein bands ranging in size from 55.3 to approximately 12.8 kDa were resolved by SDS-polyacrylamide gel electrophoresis. A 31.6-kDa protein was identified as cytochrome c1 by the presence of a covalently attached heme. A red shift in the alpha-absorbance band of the cytochrome c1 absolute absorbance spectrum, difference absorbance spectrum, and pyridine ferrohemochrome absorbance spectrum suggested that the heme prosthetic group of C. fasciculata cytochrome c1 is bound to the apoprotein through only one thioether bond. A fragment of the cytochrome c1 gene was amplified from C. fasciculata, Trypanosoma brucei, Leishmania tarentolae, and Bodo caudatus. The deduced heme binding site sequence of each of these kinetoplastid species, Phe-Ala-Pro-Cys-His, contains a phenylalanine rather that a cysteine at the first position so that only one thioether bond can be formed between heme and apoprotein. This phenylalanine substitution and the presence of a conserved proline in the sequence may represent compensatory changes that are necessary for optimal interaction of the cytochromes c1 with the atypical cytochromes c of these species.     

NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.     

Solution structure of mitochondrial cytochrome c. II. 1H nuclear magnetic resonance of ferrocytochrome c

The 1H nuclear magnetic resonance spectrum of tuna ferrocytochrome c has been studied and the resonances of all 49 amino acid methyl groups have been assigned to specific absorption lines. In comparison with resonance assignments in the ferricytochrome c spectrum, the secondary shifts of resonances of ferrocytochrome c are smaller and the identification of characteristic spin-systems from comparison of spectra from homologous proteins more difficult. For this reason, two-dimensional nuclear magnetic resonance exchange correlated spectroscopy has been used to correlate the assigned resonances of tuna ferricytochrome c with previously unassigned resonances of tuna ferrocytochrome c.     

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.     

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aromatic amino acids

The aromatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. 33 resonances arising from 39 aroumatic protons of ferrocytochrome c, and 18 resonances arising from 27 aromatic protons of ferricytochrome c have been assigned.     

Relation of the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin

The relationship between the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin was investigated. The rates of reduction of phosvitin-bound ferricytochrome c by cytochrome b2, ascorbate and the superoxide radical generated by xanthine oxidase wer repressed where the binding ratio was less than half the maximum, but at higher ratios they were restored gradually with increase in the ratio. The affinity of cytochrome b2 for cytochrome c was not affected by binding of cytochrome c to phosvitin. The redox potential of the bond form was lower than that of the free form and only decreased with decrease in the ratio. The conformatin around the heme moiety and the electronic structure of the heme group of bound ferricytochrome c were similar to those of free ferricytochrome c, but the conformational stability in the vicinity of the prosthetic group was related to the binding ratio as ratios above half the maximum and was well correlated with the reduction rate. Since the binding of cytochrome c to phosvitin is much stronger at binding ratios below half the maximum, these results suggest that this binding strength exclusively affects the conformational flexibility of the heme crevice in the cytochrome molecule, thus altering the reduction rate.     

13C and proton NMR studies of horse cytochrome c. Systematic assignment of methyl and methine resonances in both oxidation states.

The CHn groups in the aliphatic side chains of horse cytochrome c have been characterized according to the chemical shifts of both 13C-NMR and 1H-NMR signals, their temperature dependence and the number of attached protons, n. The primary assignments of resonances from the 55 side-chain methyl and the 27 methine groups were obtained directly for the oxidised and the reduced forms. Specific assignments of the 13C resonances were obtained through shift-correlation experiments and comparison with earlier 1H-NMR studies, by further measurements of proton-proton interactions, or by elimination. Comparison of the paramagnetic shifts of carbon and protons indicates a small redox-related change of conformation in the vicinity of Trp59 and a significant expansion of the protein over 30-50 degrees C.     

Proton NMR spectroscopy of cytochrome c-554 from Alcaligenes faecalis

Cytochrome c-554 from the bacterium Alcaligenes faecalis (ATCC 8750) is a respiratory electron-transport protein homologous to other members of the cytochrome c family. Its structure has been studied by 1H NMR spectroscopy in both the ferric and ferrous states. The ferric spectrum is characterized by downfield hyperfine-shifted heme methyl resonances at 46.25, 43.60, 38.40, and 36.73 ppm (25 degrees C, pH 7.1). Chemical shifts of these resonances change with temperature opposite to expectations derived from Curie's law. The pH behavior of the hyperfine-shifted resonances titrates with a pK of 6.3 that has been interpreted as due to ionization of a heme propionate. In the ferrous state, heme methyl, meso, and thioether bridge resonances have been observed and assigned. All aromatic proteins have been assigned according to the side chain of origin, and the structural environment about the sole tryptophan residue has been examined. The electron-transfer rate between ferric and ferrous forms has been estimated to be on the order of 3 X 10(8) M-1 s-1, which is the largest such self-exchange rate yet observed for a cytochrome.     

A study of the electron transfer properties of the heme undecapeptide from cytochrome c by 1H nmr spectroscopy

Nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the heme undecapeptide from cytochrome c. Assignments of resonances to specific residues have been made based on spin decoupling, redox titration, and the pH and temperature dependence of resonance lines. An outline structure is presented based on the assignments, secondary shift data, and the x-ray crystal structure of cytochrome c. An equation is derived to relate the width of an nmr line during a redox titration to the percentage of each oxidation state. Using this equation the self-exchange rate constant for electron transfer for the heme peptide is 1.3 x 10(7) M-1 sec-1 at 330 degrees K. Discussion of the self-exchange rate constants of cytochrome c, cytochrome c3, and cytochrome c551 is related to this constant for the heme undecapeptide.