Axial Histidine Research Articles

Cloning and sequence analysis of the gene encoding Methylophilus methylotrophus cytochrome c″, a unique protein with a perpendicular orientation of the histidinyl ligands

Cytochrome c″ from Methylophilus methylotrophus is an unusual monohaem protein that undergoes a major redox-linked spin-state transition: one of the two axial histidines bound to the iron in the oxidised form is detached upon reduction and a proton is taken up. A 3.5-kb DNA fragment, containing the gene encoding cytochrome c″ ( cycA), has been cloned and sequenced. The cytochrome c″ gene codes for a pre-protein with a typical prokaryotic 20-residue signal sequence, suggesting that the protein is synthesised as a precursor which is processed during its secretion into the periplasm. The C-terminus of cytochrome c″ has homology with the corresponding region of an oxygen-binding haem protein (SHP) from phototrophically grown Rhodobacter sphaeroides. SHP is similar in size and in the location of its haem-binding site. Immediately downstream from cytochrome c″ a second open reading frame (ORF) codes for a 23-kDa protein with similarity to the cytochrome b-type subunit of Ni-Fe hydrogenase. The possibility of coordinated expression of cycA and this ORF is discussed.

Hemoprotein models based on a covalent helix-heme-helix sandwich. 3. Coordination properties, reactivity and catalytic application of Fe(III)- and Fe(II)-mimochrome I

The coordination state of Fe(III)- and Fe(II)-mimochrome I, a covalent peptide-deuteroheme sandwich involving two nonapeptides bearing a histidine residue in a central position, was studied by UV-visible, EPR, and resonance Raman spectroscopy. The ferric and ferrous states of this new iron species mainly exist, at pH 7, in a low-spin hexacoordinate form with two axial histidine ligands coming from the peptide chains. A minor amount of high-spin form for the ferric state is also present at pH 7. However, it is mainly high-spin at pH 2 or in DMSO. Fe(II)-mimochrome I binds CO with an affinity comparable to that of myoglobin and hemoglobin. Fe(III)-mimochrome I reacts with alkylhydroxylamine and arylhydrazines, leading to the corresponding Fe(II)-nitrosoalkyl and Fe(III)-σ-aryl complexes, respectively. These reactions were greatly dependent on the solvent used and on the pH, and were much slower than the corresponding reactions performed by deuterohemin in the presence of excess imidazole. All these results indicate that the reactivity of iron-mimochrome I is controlled by the binding of the peptide chains to the iron. The reactivity shown by this complex at neutral pH is intermediate between that observed for iron porphyrins in the presence of excess imidazole and that of hemoproteins characterized by a strong bis-histidine axial coordination, such as cytochrome b5. Fe(III)-mimochrome I is able to catalyze styrene epoxidation by using a [Fe(III)-mimochrome I]/[H2O2]/[stryrene] ratio of 1 : 10 : 2000 in phosphate buffer solution (pH 7.2) containing 2% CTAB both under strictly anaerobic conditions and in the presence of oxygen, at 0 °C.

Structural and functional roles of heme binding module in globin proteins: identification of the segment regulating the heme binding structure

To investigate structural and functional significance of a newly proposed structural unit in globins, the “heme binding module”, we synthesized a “heme binding module”-substituted chimeric globin and characterized its function and structure. In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin α-subunit, plays a key role in constructing the heme proximal structure in globins. The replacement of the heme binding module in myoglobin with that of hemoglobin α-subunit converted the absorption spectra into that of the α-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement. The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the α-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site. The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins.

Modeling Electron Transfer in Biochemistry: A Quantum Chemical Study of Charge Separation in Rhodobacter sphaeroides and Photosystem II

High-level quantum chemical methods (hybrid density-functional type) are applied to the light-driven charge-separation process in the photosynthetic reaction centers of both bacteria and photosystem II in green plants. Structural information on the bacterial system provides the basis for choosing the models used in the calculations. The energetics of the electron transfer from the (bacterio)chlorophyll to the quinone are calculated as well as those of the intermediate step involving the (bacterio)pheophytin. The surrounding protein is treated as a dielectric medium, and the cavities around the solute molecules are determined by isodensity surfaces. The dielectric effects on the charge-separation processes are calculated to be as large as 50 kcal/mol. It is shown that hydrogen bonding between the chromophores and certain peptide residues as well as the axial histidine ligand on the (bacterio)chlorophylls contributes substantially to the energetics. Good agreement with experimentally estimated driving force...

PH Dependence of Structural and Functional Properties of Oxidized Cytochrome c" from Methylophilus methylotrophus

Cytochrome c" from Methylophilus methylotrophus is an unusual monoheme protein that undergoes a major redox-linked change in the heme arrangement: one of the two axial histidines bound to the iron in the oxidized form is detached upon reduction and a proton is taken up. The kinetics of reduction by sodium dithionite and the spectroscopic properties of the oxidized cytochrome c" have been investigated over the pH range between 1.4 and 10.0. The rate of reduction displays proton-linked transitions of pKa congruent with 5.5 and 2.4, and a spectroscopic transition with a pKa congruent with 2.4 is also observed. The protein displays a complete reversibility after exposure to low pH, and both electronic absorption and resonance Raman spectroscopic properties suggest that the transition at lower pH brings about a drastic change in the heme coordination geometry. Circular dichroism spectra indicate that over the same proton-linked transition, the protein undergoes a marked decrease (approximately 60%) of the alpha-helical content toward a random coil arrangement, which is recovered upon increasing the ionic strength. The structural change at low pH is linked to a concerted two-proton transition, suggesting the detachment and protonation of axial histidine(s). Such kinetic and spectroscopic features along with the remarkable capacity of this protein to recover its native structure after exposure to extremely low pH values makes it a promising model for studying folding processes and stability in heme proteins.

Further characterization of the two tetraheme cytochromes c3 from Desulfovibrio africanus: nucleotide sequences, EPR spectroscopy and biological activity

The genes encoding the basic and acidic tetraheme cytochromes c 3 from Desulfovibrio africanus have been sequenced. The corresponding amino acid sequences of the basic and acidic cytochromes c 3 indicate that the mature proteins consist of a single polypeptide chain of 117 and 103 residues, respectively. Their molecular masses, 15 102 and 13 742 Da, respectively, determined by mass spectrometry, are in perfect agreement with those calculated from their amino acid sequences. Both D. africanus cytochromes c 3 are synthesized as precursor proteins with signal peptides of 23 and 24 residues for the basic and acidic cytochromes, respectively. These cytochromes c 3 exhibit the main structural features of the cytochrome c 3 family and contain the 16 strictly conserved cysteine+histidine residues directly involved in the heme binding sites. The D. africanus acidic cytochrome c 3 differs from all the other homologous cytochromes by its low content of basic residues and its distribution of charged residues in the amino acid sequence. The presence of four hemes per molecule was confirmed by EPR spectroscopy in both cytochromes c 3. The g-value analysis suggests that in both cytochromes, the angle between imidazole planes of the axial histidine ligands is close to 90° for one heme and much lower for the three others. Moreover, an unusually high exchange interaction (∼10 −2 cm −1) was evidenced between the highest potential heme (−90 mV) and one of the low potential hemes in the basic cytochrome c 3. The reactivity of D. africanus cytochromes c 3 with heterologous [NiFe] and [Fe] hydrogenases was investigated. Only the basic one interacts with the two types of hydrogenase to achieve efficient electron transfer, whereas the acidic cytochrome c 3 exchanges electrons specifically with the basic cytochrome c 3. The difference in the specificity of the two D. africanus cytochromes c 3 has been correlated with their highly different content of basic and acidic residues.

Paramagnetic NMR shifts in cyanoferricytochrome c: Investigation of thermal stability and deviations from Curie law behaviour

The paramagnetic shifts of 13C nuclei positioned α to the haem in cyanoferricytochrome c are reported and analysed in terms of molecular orbitals based on D 4h symmetry with a rhombic perturbation. The temperature dependence of the Fermi contact and dipolar shifts of the haem and axial histidine ligand show deviations from Curie Law behaviour which are explained by a Boltzmann distribution between partially filled 3e( π) molecular orbitals and the ground and first excited state Kramers doublets. The comprehensive explanation of the temperature dependence of the paramagnetic shifts leads to the conclusion that there is no detectable temperature dependence of the haem orientation or that of the His ligand orientation. This work also provides evidence for the role of the axial His ligand in determining the orientation of the magnetic z-axis.

Demonstration of the Role of Scission of the Proximal Histidine−Iron Bond in the Activation of Soluble Guanylyl Cyclase through Metalloporphyrin Substitution Studies

Activation of soluble guanylyl cyclase (sGC) by NO correlates with scission of the proximal iron−histidine bond, as demonstrated by the application of electronic absorption and resonance Raman spectroscopy to the study of metalloporphyrin-substituted enzymes. The non-native metalloporphyrins, Mn(II)PPIX and Co(II)PPIX, can be introduced into heme-deficient sGC forming five-coordinate complexes. The similarity among Mn(II)sGC, Co(II)sGC, and the corresponding metalloporphyrin-substituted derivatives of Mb and Hb provides confirming evidence for the presence of an axial histidine ligand in sGC. Upon addition of NO, Mn(II)sGC forms a six-coordinate species with the histidine ligand still bound to the Mn, and the enzyme is not activated. In contrast, the Co(II)sGC(NO) adduct is five-coordinate and the enzyme is activated. These data imply that the activated state of sGC is attained when the proximal histidine−metal bond is broken.

Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

The chemical shifts of several 13C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D4h symmetry. The additional contributions to the paramagnetic shifts of 13C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental 13C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from 13C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on 13C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from 13C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available.

Molecular Dynamics of Microperoxidases in Aqueous and Nonaqueous Solutions

We report an extended molecular dynamics study of the heme-containing peptide “microperoxidase” (MP), a model system of several heme-proteins. The most commonly studied forms of MP, the octa- and the undecapeptide, are studied both in pure water conditions and in mixed solvent (80% methanol and 20% water) conditions that stabilize the monomeric form. The equilibrium structures and their stability are visualized by graphical tools and analyzed in terms of the molecular shape, the vibrational amplitudes, and torsional angles along the backbone. The obtained structures are used to confirm the experimental data on aggregation of Urry and Pettegrew7,8 by using simple geometric arguments based on the possible assemblies of several monomeric units in space. We have investigated in detail the H-bond network and the influence on the heme conformational properties. We have found that heme interacts with the peptide through H-bonds formed by the propionates, the axial histidine, and different partners of the peptidic chain. These interactions are regulated by the degree of exposure of HIS to the solvent. These results permit us to predict that the undecapeptide and the octapeptide can be good model systems for horseradish peroxidase and cytochrome c‘, respectively.

Heme-Peptide Models for Hemoproteins. 1. Solution Chemistry of N-Acetylmicroperoxidase-8.

An improved method for the preparation of the heme octapeptide acetyl-MP8, obtained by proteolysis of horse heart cytochrome c, is described. AcMP8 obeys Beer's law at pH 7.0 in aqueous solution up to a concentration of 3 x 10(-)(5) M. The self-association constant measured at 25 degrees C (log K(D) = 4.04) is an order of magnitude lower than that for MP8, reflecting the role of the N-acetyl protecting group in abolishing intermolecular coordination. However, AcMP8 does form pi-stacked dimers in aqueous solution with increasing ionic strength. A more weakly packed pi-pi dimer reaches a maximum abundance at approximately 3 M ionic strength, but a more tightly packed dimer is favored at &mgr; > 3 M. An equilibrium model based on charge neutralization by specific binding of Na(+) ions gives a total molecular charge of 3- for AcMP8 at pH 7.0 and a self-association constant log K(D) = 4.20. AcMP8 exhibits six spectroscopically active pH-dependent transitions. The Glu-21 c-terminal carboxylate binds to the heme iron at low pH (pK(a) = 2.1) but is substituted by His-18 (pK(a) = 3.12) as the pH increases. The two heme propanoic acid substituents ionize with pK(a)'s of 4.95 and 6.1. This is followed by ionization of iron-bound water with a pK(a) = 9.59, DeltaH = 48 +/- 1 kJ mol(-)(1), and DeltaS = -22 +/- 3 J K(-)(1) mol(-)(1). The electronic spectra indicate that AcMP8 is predominantly in the S = (5)/(2) state at pH 7.0, while the hydroxo complex at pH 10.5 corresponds to an equilibrium mixture of S = (5)/(2) and S = (1)/(2) states at 25 degrees C. In the final transition, His-18 ionizes to form the S = (1)/(2) histidinate complex with a pK(a) of 12.71. AcMP8 is relatively stable under alkaline conditions, dimerizing slowly at high pH (k = 2.59 +/- 0.14 M(-)(1) s(-)(1)) to form a high-spin &mgr;-oxo-bridged species. The pH-dependent behavior of AcMP8 in the presence of excess 3-cyanopyridine, however, is markedly different. At low pH, AcMP8 simultaneously binds the exogenous ligand and the Glu-21 c-terminal carboxylate with a pK(a) < 2. His-18 replaces the carboxylate ligand at higher pH (pK(a) = 2.60), and both heme propanoic acid groups ionize with a mean pK(a) = 5.10. Unlike AcMP8.OH(-), the axial histidine of the 3-CNPy complex ionizes at near neutral pH (pK(a) = 7.83), prior to being replaced by OH(-) (pK(a) = 10.13). The sixth transition in the AcMP8/3-CNPy system produces the bis(hydroxo) complex (pK(a) > 13).

Three-dimensional structure of galactose-1-phosphate uridylyltransferase from Escherichia coli at 1.8 A resolution.

Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDP-galactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multiple isomorphous replacement and electron density modification techniques and has been refined to 1.8 A resolution. Enzyme subunits consist of a single domain with the topology of a "half-barrel". The barrel staves are formed by nine strands of antiparallel beta-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the half-barrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, S gamma of a cysteine is modified by beta-mercaptoethanol, which prevents the alpha-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Leu54, Ohe75, Asn77, Asp78, Phe79, and Val108. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations.

Carbon-13 NMR studies of the influence of axial ligand orientation on haem electronic structure

Three-quarters of the carbon-13 resonances of nuclei attached to the four haems of Desulfovibrio uulgaris ferricytochrome c 3 are assigned. Preliminary analysis of their Fermi contact interactions shows that the shifts are directly related to the orientation of both of the axial histidine ligands in each case and the approach can therefore be used to obtain structural information in other cytochromes with bis-histidinyl coordination. The implications for the control of redox potential in cytochromes are discussed.

Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa 3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a 3) and copper (Cu B). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe 3+-Cu +center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa 3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe 3+ -Cu 2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa 3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa 3. This suggests that the binuclear centers of the two enzymes are quite similar.

Electric Field and Conformational Effects of Cytochrome c and Solvent on Cytochrome c Peroxidase Studied by High-Resolution Fluorescence Spectroscopy

Electronic spectra of mesoporphyrin-substituted yeast cytochrome c peroxidase (MP-CcP) were measured as a function of pH, ionic strength, and binding of cytochrome c (cyt c) by fluorescence line narrowing (FLN) spectroscopy at 5 K. The FLN spectra provided information about the vibrational structure of the first excited singlet state of MP-CcP, the various tautomeric forms of mesoporphyrin, and the positions and widths of their 0,0 bands. The composite 0,0 band of MP-CcP at pH 6 could be resolved into three components with peak positions at 16,046, 16,103, and 16,203 cm-1. MP-CcP at pH 8 could be analyzed using two components with peak positions at 16,048 and 16,193 cm-1. The disappearance of the 16,103-cm-1 component at alkaline pH suggests that it is due to a "chemical substate" arising from protonation of His52 in the distal side of the porphyrin. Computer simulations of the electrostatic field that CcP imposes on its porphyrin show that, in the presence of charged axial histidines His52 and His175, the electrostatic field at porphyrin nitrogens increases, especially along the normal to the heme by about 200 mV/A. Electric field effects may account for pH-dependent spectral shifts of the 0,0 positions of the resolved components, although hydrogen bonding may also affect these positions. On the other hand, the peak position of the components was not affected by ionic strength or binding of cyt c, implying that the electrostatic field of the heme pocket of MP-CcP remains unchanged. Indeed, computed changes in ionic strength of the solvent show no modification of the electrostatic field at the porphyrin. The only detectable effect of ionic strength and binding of cyt c to MP-CcP is on the relative contributions of the components, suggesting some rearrangements in the vicinity of the heme. Finally, shifts in the position of the vibrational lines for MP-CcP components indicate either that the tautomers have different vibrational frequencies due to the nonsymmetry of the porphyrin and/or that tautomers experience various distortions. Comparison of the vibrational spectrum of the first excited singlet state of mesoporphyrin in CcP and horseradish peroxidase also suggests that the heme pocket in the two peroxidases provides different steric restrictions.

Hydropathy and Molar Volume Constraints on Combinatorial Mutants of the Photosynthetic Reaction Center

Combinatorial cassette mutagenesis was used to substitute randomly nine amino acid residues in the vicinity of the active branch monomeric bacteriochlorophyll in the photosynthetic reaction center of Rhodobacter capsulatus. The bacteriochlorophyll environment was targeted because of the potential role of this cofactor in the initial charge separation event of photosynthesis. Mutants with perturbed binding and which have altered energy levels of this chromophore, would be useful for electron transfer studies. Four sites in the M-subunit D-helix and five sites in the L-subunit cd-helix, including residue L153, the axial histidine ligand to the bacteriochlorophyll, were randomly substituted. The cd and D-helix regions were mutagenized independently of each other and simultaneously, resulting in three libraries of mutants. Digital imaging spectroscopy was used to screen photosynthetically selected mutants for ground state absorption spectra in the visible and near-infrared. The functional mutants of each library have distinct spectroscopic characteristics. One unusual spectral phenotype with an absorbance band at 825 nm occurred only in the nine-site library, with a frequency of about one out of 10 6 mutants. A mutant with an 825 nm band also has an unusual light-minus-dark difference spectra, which does not show a blue shift of the 800 nm absorption band. The mean molar volume and hydropathy of the substitutions occurring in functional mutants are biased towards the mean values of a random dope to various extents, indicating different stringencies at each site for these physicochemical properties.

Assignment of heme resonances in the 1H NMR spectrum of oxidized Desulfovibrio vulgaris (Hildenborough) cytochrome c3

A combination of 2D COSY and NOESY experiments and 1D nuclear Overhauser effect measurements have allowed an extensive assignment of the hyperfine-shifted heme resonances in the downfield region of the 1H NMR spectrum of the fully oxidized tetraheme cytochrome c 3 from Desulfovibrio vulgaris, Hildenborough. Fifteen of the sixteen heme methyl groups were identified and assigned, some on a tentative basis. Most of the one-proton paramagnetic resonances were demonstrated to arise from the α-CH 2 groups of the heme propionate side chains, and from the β-CH 2 groups of the iron-binding axial histidines. Assignment of methyl resonances to individual hemes was achieved by reference to previous calculations of electron density distributions on the four hemes of the highly homologous Miyazaki protein obtained by NMR data ( Biochemistry, 29 (1990) 2257). The methyl hyperfine shift patterns are apparently different for the individual hemes. Despite the complexity of the system due to interheme interactions, we have determined that these patterns appear to be influenced by the orientation of the imidazole planes of the iron-binding axial histidines, as found for simpler monoheme proteins.

Involvement of a labile axial histidine in coupling electron and proton transfer in Methylophilus methylotrophus cytochrome c''.

Methylophilus methylotrophus cytochrome c'' is an unusual monohaem protein (15 kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277-286]. The midpoint redox potential of cytochrome c" was measured over the pH range 4-10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pKa10 = 6.4, pKa1r = 5.4 and pKa2r = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pKar was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKa0 too low to be measured. It is shown that M. methylotrophus cytochrome c" is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed.

Spectroscopic studies of Rhodobacter capsulatus cytochrome c′ in the isolated state and in intact cells

Ferricytochrome c′ from Rhodobacter capsulatus was investigated by 1H-NMR, EPR and optical spectroscopies. A haem-linked ionisation, occurring with a p K a of 8.4 at 25°C, was observed and assigned to the ionisation of the axial histidine ligand by comparison with data for related proteins. At pH values below this p K a the spin-state of the haem Fe 3+ is shown to be a quantum mechanically admixed S= 3 2 , 5 2 state. Above the p K a the Fe 3+ is high-spin. EPR studies of intact cells grown photoheterotrophically reveal that in situ cytochrome c′ exists largely in the ferrous state. Upon the addition of [Fe(CN) 6] 3+ the protein becomes oxidised and EPR spectra reveal that the Fe 3+ spin-state is a quantum mechanically admixed S= 3 2 , 5 2 state. These data indicate that the unusual spin-state of ferricytochrome c′ is not a consequence of changes to the protein on its isolation, as had been suggested previously. They also indicate that in situ cytochrome c′ is located in an environment with a pH < 7.

Spectroscopic studies of Rhodobacter capsulatus cytochrome c′ in the isolated state and in intact cells

Ferricytochrome c′ from Rhodobacter capsulatus was investigated by 1H-NMR, EPR and optical spectroscopies. A haem-linked ionisation, occurring with a p K a of 8.4 at 25°C, was observed and assigned to the ionisation of the axial histidine ligand by comparison with data for related proteins. At pH values below this p K a the spin-state of the haem Fe 3+ is shown to be a quantum mechanically admixed S = 3/2, 5/2 state. Above the p K a the Fe 3+ is high-spin. EPR studies of intact cells grown photoheterotrophically reveal that in situ cytochrome c′ exists largely in the ferrous state. Upon the addition of [Fe(CN) 6] 3+ the protein becomes oxidised and EPR spectra reveal that the Fe 3+ spin-state is a quantum mechanically admixed S = 3/2, 5/2 state. These data indicate that the unusual spin-state of ferricytochrome c′ is not a consequence of changes to the protein on its isolation, as had been suggested previously. They also indicate that in situ cytochrome c′ is located in an environment with a pH< 7.