Geoffrey R. Moore

Mössbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe Mössbauer spectroscopy. The Mössbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.

Comparison of the solution and crystal structures of mitochondrial cytochrome c: Analysis of paramagnetic shifts in the nuclear magnetic resonance spectrum of ferricytochrome c

The two accompanying papers describe the assignment of methyl-containing spin-systems in the 1H nuclear magnetic resonance spectra of tuna ferricytochrome c and tuna ferrocytochrome c. At present, 104 resonances from 208 C-H protons are assigned in both oxidation states. In this paper, the difference in chemical shift of a resonance between the two oxidation states is used together with a dipolar model of the unpaired electron spin of ferricytochrome c to compare the structure of cytochrome c in solution with three high-resolution structures of cytochrome c obtained by X-ray diffraction in single crystals. The overall protein fold and the positions of most of the haem-packing residues are shown to be invariant between the crystal and solution. However, three regions of the protein, at the C terminus, around the haem propionic acid groups and at the haem crevice near thioether-2, are found to undergo conformational changes on the removal of crystal packing constraints.

Structural basis for the variation in redox potential of cytochromes

One of the aims of the intensive work undertaken to explore protein structure is to explain variations in the properties of proteins at a structural level. In the case of haem-containing electron transfer proteins one basic property to be understood is the redox potential. The redox potential of cytochromes range from t380 mV to -500 mV [l] . Recently much iuformation has been obtained about the structure of cytochromes [2-51. Here we correlate this structural information with redox potential data using the known redox potentials of model complexes in aqueous solution [6-81 to guide us to the main factors which are likely to be important. These factors are as follows:

A spectroscopic investigation of the structure and redox properties of Escherichia coli cytochrome b-562

The six-coordinate monohaem ferricytochrome b-562 from Escherichia coli exhibits two haem-linked pH-dependent transitions detected by NMR and optical spectroscopy. Only one of these transitions, that of the Fe(III)-coordinated His-102, is detected by EPR and MCD; the ionisation of a haem propionate is not. Both ionisations are redox-state-dependent and the midpoint redox potential of the protein is markedly pH-dependent. Over the pH range 5.0 to 8.5 the potential drops from 260 mV to 110 mV and at least five single proton ionisations are responsible for this. In addition to the two spectroscopically identified ferricytochrome ionisations, there are at least three unidentified ionisations, two of which occur in the ferrous protein. From a consideration of the X-ray structure, together with NMR data, it seems probable that at least one of these ionisations involves an amino acid carboxylate. The X-ray structure also suggests that the relatively low pKa of His-102 is a result of its proximity to Arg-98. However, an appreciable interaction between these groups requires that the solution conformation differs slightly from the X-ray structure. The fast rate of electron self-exchange, over 4 X 10(6) M-1 X s-1 at 315 K and pH* 7, may be a reflection of the fact that, as shown by the X-ray structure, a large amount of the haem and axial histidine ligand are exposed at the molecular surface with an asymmetric distribution of charged groups surrounding them.

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

Abstract Several cytochromes c 2 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 (p K o ) and one in the reduced form (p K r ). These cytochromes fall into one of two groups according to the degree of separation of p K o and p K r . In group A, represented here by the Rhodomicrobium vannielii cytochrome c 2 , 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 c 2 and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the p K 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 p K for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c 2 , the separation between p K o and p K 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 c 2 /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 c 2 . Using Coulombs law, it was found that the energy required to separate p K o and p K r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.

Nuclear magnetic resonance studies of metal substituted horse cytochrome c

Abstract The proton nuclear magnetic resonance spectra of various metal substituted derivatives of horse cytochrome c have been studied and compared to the spectra of native cytochrome c . The proteins studied were the cobalt(III), copper(II), iron(II), iron(III), manganese(III), nickel(II), and zinc(II) derivatives. Spectra of the diamagnetic cobalt(III), iron(II), and zinc(II) proteins were well-resolved and specific resonance assignments were made. All three proteins possessed a methionine ligand to the metal. The spectrum of cobalt(III) cytochrome c was investigated in some detail as this protein was used as a diagmagnetic control for iron(III) cytochrome c . Comparison of the spectra of cobalt(III) and iron(II) cytochromes c revealed that their conformations were very similar but the following conclusion could be made; the oxidation of cytochrome c is accompanied by a small conformation change.

Correlation of proton chemical shifts in proteins using two-dimensional exchange correlated spectroscopy

The full utilization of the information contained in protein NMR spectra requires the assignment of resonances to specific residues. Many one-dimensional and twodimensional NMR experiments can be used to give assignments to amino acid type (1-5) but, with the exception of relatively small proteins, assignments to specific residues can only be obtained by the use of chemical modification or from prior knowledge of the three-dimensional structure of the protein. In some cases, such considerations lead to a considerable simplification of the assignment procedure. A simplification is also possible where the study requires the protein to be compared in two or more different states, such as the native or nonnative states (6, 7) or the ligand-free and ligand-bound states (8). In such cases, provided the spectrum of one of the states has been previously characterized and there is exchange between the two states, the spectrum of the second state can be assigned. Tuna cytochrome c is a heme-containing electron transfer protein of 103 amino acids that may exist in either a diamagnetic oxidation state, ferrocytochrome c or a paramagnetic oxidation state, ferricytochrome c. The iron is low-spin in both oxidation states, and in ferricytochrome c it acts as a shift probe with a highly anisotropic magnetic moment. Consequently, the NMR spectra of ferricytochrome c and ferrocytochrome care very different (9). Because of the paramagnetism of ferricytochrome c, its ‘H NMR spectrum is relatively well resolved, and this has allowed resonances of approximately 45% of its carbon-bonded protons to be specifically assigned using a combination of one-dimensional NMR techniques and specific chemical modification (IO, 1Z). In contrast, the ‘H NMR spectrum of ferrocytochrome c has a considerably smaller chemical-shift dispersion, which allows fewer selective double-resonance experiments, and consequently there are fewer specific assignments available. In the present communication, making use of the assignments of ferricytochrome c, we demonstrate the utility of chemical exchange NMR to assign the corresponding resonances of ferrocytochrome c. An equimolar mixture of ferricytochrome c (CIII) and ferrocytochrome c (CII) undergoes electron exchange according to km *c111 + CII z=t *c11 + CIII. ill

Biological Electron Transfer: The Structure, Dynamics and Reactivity of Cytochrome c

Abstract Cytochrome c is the best characterized member of a group of over 50 metallo- and flavo-proteins which constitute the mitochondrial respiratory electron transport chain. It is a comparatively small (103–111 amino acid residues), stable, water-soluble protein containing a single haem prosthetic group which cycles between the Fe(II) and Fe(III) states in the course of its electron transfer reactions. In a recent Faraday Society Discussion we presented a paper1 which concluded with the following paragraphs:

The effect of iron-hexacyanide binding on the determination of redox potentials of cytochromes and copper proteins

The midpoint redox potentials of Pseudomonas aeruginosa cytochrome c-551 and Rhodopseudomonas viridis cytochrome c2 were measured as a function of pH in the presence of Euglena cytochrome c-558 and the results compared with those obtained in the presence of ferro-ferricyanide. The pattern of pH dependence observed for the two bacterial cytochromes was the same whether it was measured by equilibrium with another redox protein or with the inorganic redox couple. Thus, the pH dependence of redox potential is not a consequence of pH-dependent ligand binding. The midpoint potential of Ps. aeruginosa azurin was measured as a function of pH using both ferro-ferricyanide mixtures and redox equilibrium with horse cytochrome c or Rhodopseudomonas capsulata cytochrome c2. In this case also the pattern of pH dependence obtained did not vary with the redox system used and it closely resembled that of Ps. aeruginosa cytochrome c-551. This is consistent with the observation that the equilibrium between cytochrome c-551 and azurin is relatively independent of pH. An equation was derived which described ph-dependent ligand binding and which can produce theoretical curves to fit the experimental pH dependence of redox potential for both cytochrome and azurin. However, the pronounced effect on such curves produced by varying the ligand association constants, and the insensitivity of the experimental data to changes in ionic strength, suggest that ligand binding effects do not account for the pH dependence of redox potential.

Nuclear magnetic resonance studies of the phenylalanine residues of eukaryotic cytochrome c

The resonances of Phe 82 and Phe 10 in the nuclear magnetic resonance spectra of horse cytochrome c are reassigned using nuclear Overhauser enhancements. The reassignments provide new information about the oxidation state linked conformation change of cytochrome c. The region of the protein now known to be affected by the change extends to the part of the protein close to Phe 10.