Characterization of the reaction between ferrocytochrome c and cytochrome c oxidase.

The reaction between cytochrome c oxidase and ferrocytochrome c has been investigated by the stopped-flow method. It has been found that only one electron acceptor, a heme group, in the oxidase is rapidly reduced by cytochrome c. The presence of N3- does not affect the reduction of the acceptor, which supports the hypothesis that this is identical with cytochrome a. The results are consistent with the existence of a simple equilibrium between cytochrome a and cytochrome c: c-2 + a-3+ in equilibrium c-3+ + a-2+ with an equilibrium constant corresponding to an oxidation-reduction potential of cytochrome a 30 mV higher than that for cytochrome c at pH 7.4. The oxidation-reduction potential of the a-3+ /a-2+ couple, 285 mV (based on a potential of 255 mV for cytochrome c), and the optical properties of the reduced form indicate that it is identical with neither of the reduced hemes seen in potentiometric titrations. The oxidase species resulting from the rapid reduction of cytochrome a by cytochrome c is proposed to represent a metastable intermediate state which, under anaerobic conditions, eventually is transformed into a more stable state characterized by a reduced high-potential heme.     

The iron-sulfur environment in rubredoxin.

The atomic environment around the iron site in the nonheme iron sulfur protein rubredoxin was studied by the extended X-ray absorption fine structure (EXAFS) technique. Within experimental error, the Fe-S bonds in oxidized Clostridium pasteurianum rubredoxin are the same as in the analogue anion [Fe(S2-o-xyl)2]-synthesized by Holm. The average Fe-S bond length is 2.267 +/- 0.003A and the root mean square deviation about this average due to structural disorder is 0.032 + 0.013 - 0.032.     

The electronic state of heme in cytochrome oxidase II. Oxidation-reduction potential interactions and heme iron spin state behavior observed in reductive titrations.

Magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and optical absorption spectroscopies have been used to monitor the concentrations of oxidized and reduced heme and copper during stoichiometric reductive titrations of purified beef heart cytochrome oxidase. The MCD data are deconvoluted to obtain the concentrations of reduced cytochromes a and a3 during the titrations; analysis of the EPR spectra provides complementary data on the concentrations of the EPR-detectable species. For the native enzyme in the absence of exogenous ligands, cytochromes a and a3 are reduced to approximately the same extent at all points in the titration. The reduction of the EPR-detectable copper, on the other hand, initially lags the reduction of the two cytochromes but in the final stages of the titration is completely reduced prior to either cytochrome a or a3. These non-Nernstian titration results are interpreted to indicate that the primary mode of heme-heme interaction in cytochrome oxidase involves shifts in oxidation-reduction potential for each of the two cytochromes such that a change in oxidation state for one of the hemes lowers the oxidation-reduction potential of the second heme by approximately 135 mV. In these titrations high spin species are detected which account for 0.25 spin/oxidase maximally. Evidence is presented to indicate that at least some of these signals can be attributed to cytochrome a3+ which has undergone a low-spin to high-spin state transition in the course of the titration. In the presence of carbon monoxide the oxidation-reduction properties of cytochromes a and a3 are markedly altered. The a32+. CO complex is fully formed prior to reduction of either cytochrome a3+ or the EPR-detectable copper. The g = 3 EPR signal attributed to cytochrome a3+ decreases as the MCD intensity of cytochrome a2+ increases; no significant high-spin intensity is observed at any intermediate stage of reduction. We interpret these Nernstian titration results to indicate that in the presence of ligands the oxidation-reduction potential of cytochrome a relative to cytochrome a3 is determined by the oxidation-reduction state of the stabilized cytochrome a3 ligand complex; if ligand binding occurs to reduced cytochrome a3 then cytochrome a titrates with a lower potential; cytochrome a titrates with a higher potential if oxidized cytochrome a3 is stabilized by ligand binding.     

Extended X-ray absorption fine structure study of Rhodospirillum rubrum and Rhodospirillum molischianum cytochromes c': relationship between heme stereochemistry and spin state

An EXAFS study on the oxidized and reduced forms of cytochromes c' from Rhodospirillum rubrum and Rhodospirillum molischianum was performed at pH 7. The cytochromes c' have an apparent coordination number of 5 in both oxidation states. Average Fe-ligand bond lengths of 2.02 +/- 0.025 and 2.06 +/- 0.025 A are obtained in their oxidized and reduced forms, respectively. By use of suitable values for the Fe-NHis bond length and Fe out-of-plane displacement, as determined by small molecule crystallographic techniques, the Fe-Npyrrole bond lengths and the porphyrin center-to-Npyrrole distance have been estimated for cytochrome c' in both of its oxidation states. With this model, estimates of the Fe-Npyrrole bond lengths are 2.01 +/- 0.03 and 2.05 +/- 0.03 A, for the oxidized and reduced cytochromes c', respectively. The center-to-Npyrrole distance is estimated to be 1.99 +/- 0.03 A for oxidized cytochrome c' and 2.03 +/- 0.03 A for reduced cytochrome c'.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.     

Oxidation-reduction properties of Chromatium vinosum high potential iron-sulfur protein.

The oxidation-reduction properties of the high potential iron-sulfur protein (HIPIP) from Chromatium vinosum have been investigated. Both equilibrium and kinetic measurements demonstrate electron transport by HIPIP is pH independent in the pH range 7-11. The kinetics of reduction (potassium ferrocyanide, SO2, S2O42-, sodium ascorbate, and Rhodospirillum rubrum cytochrome c2) and oxidation (potassium ferricyanide and Rhodospirillium rubrum cytochrome c2) of HIPIP are reported. Based on the data obtained with different reactants and the influence of ionic strength, pH, and temperature on the kinetics of oxidation and reduction, a number of conclusions can be drawn. (1) HIPIP undergoes rapid outer-sphere electron transfer with no evidence of kinetic complexity and no indication of complex formation with various reactants. (2) The site of oxidation of reduced HIPIP has an apparent negative charge while the site of reduction of oxidized HIPIP is uncharged. (3) HIPIP appears to interact with a physiological reactant (R. rubrum cytochrome c2) at the same site as nonphysiological oxidants or reductants suggesting single minimum energy pathways for the oxidation and reduction processes. (4) Based on a comparison of the rates of oxidation and reduction with different reactants, it appears that steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants. (5) The thermodynamic activation parameters indicate that both oxidation and reduction by the iron hexacyanides are driven entropically with the enthalpic terms making no contribution to HIPIP oxidation and a small contribution to HIPIP reduction. Based on the data reported here and available structural and physical-chemical information, possible mechanisms of the oxidation and reduction of HIPIP are discussed and their relative merits analyzed. The more likely mechanisms include electron transfer via a tyrosine residue, electron transfer through a nonaqueous media to the iron-sulfur chromophore, and direct interaction between the iron-sulfur chromophore and the different oxidants and reductants.     

Potentiometric and spectroscopic properties of the cytochrome o complex of Escherichia coli

Cytochrome o purified from cell membranes of Escherichia coli shows two potentiometrically distinct species with midpoint oxidation-reduction potentials of +265 +/- 5 and +140 +/- 15 mV. The component with the higher potential reacted with carbon monoxide and so likely is the oxygen-reacting heme of the cytochrome o complex. It appears to be responsible for the absorption maximum at 564 nm in reduced minus oxidized difference spectra measured at 77 K. The midpoint potential of the other component was sensitive to oxidation by ferricyanide. This latter component had an absorption maximum at about 554 nm. The inhibitor 2-heptyl-4-hydroxyquinoline N-oxide inhibited reoxidation of reduced cytochrome o by oxygen and modified the spectroscopic behaviour of the 564 nm component. The ratio of the heights of the maxima in the alpha-band region of the absorption spectrum differed in cytochrome o prepared from cloned material from that found in cytochrome o from noncloned sources, in spite of the similar polypeptide compositions of the two preparations.     

High and low reduction potential 4Fe-4S clusters in Azotobacter vinelandii (4Fe-4S) 2ferredoxin I. Influence of the polypeptide on the reduction potentials.

Azotobacter vinelandii (4Fe-4S)2 ferredoxin I (Fd I) is an electron transfer protein with Mr equals 14,500 and Eo equals -420 mv. It exhibits and EPR signal of g equals 2.01 in its isolated form. This resonance is almost identical with the signal that originates from a "super-oxidized" state of the 4Fe-4S cluster of potassium ferricyanide-treated Clostridium ferredoxin. A cluster that exhibits this EPR signal at g equals 2.01 is in the same formal oxidation state as the cluster in oxidized Chromatium High-Potential-Iron-Protein (HiPIP). On photoreduction of Fd I with spinach chloroplast fragments, the resonance at g equals 2.01 vanishes and no EPR signal is observed. This EPR behavior is analogous to that of reduced HiPIP, which also fails to exhibit an EPR spectrum. These characteristics suggest that a cluster in A. vinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP. Quantitative EPR and stoichoimetry studies showed that only one 4Fe-4S cluster in this (4Fe-4S)2 ferredoxin is reduced. Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy. This indicates that a cluster in Fd I shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic. The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP. The midpoint reduction potential of this cluster is approximately +340 mv. A. vinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (approximately +340 mv) and a low potential cluster (-420 mv). Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster in Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are approximately 760 mv different.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

On the question of interheme electron transfer in the chloroplast cytochrome b6 in situ

The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation. (i) The two hemes of the cytochrome ba and bp, have similar (delta Em less than or equal to 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, ba, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 +/- 0.09 heme/600 Chl) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme ba was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme ba as specified by the Q cycle model. (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 approximately 30 ms) when heme ba was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme ba indicates that the oxidation of ba and bp may be cooperative.     

Oxidation-reduction properties of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Native Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase contains a [4Fe-4S] cluster in the diamagnetic (+2) state. The cluster is essential for catalytic function, even though amidotransferase does not catalyze a redox reaction. The ability of the Fe-S cluster to undergo oxidation and reduction reactions and the consequences of changes in the redox state of the cluster for enzyme activity were studied. Treatment of the enzyme with oxidants resulted in either no reaction or complete dissolution of the Fe-S cluster and loss of activity. A stable +3 oxidation state was not detected. A small amount of paramagnetic species, probably an oxidized 3Fe cluster, was formed transiently during oxidation. The native cluster was poorly reduced by dithionite, but it could be readily reduced to the +1 state by photoreduction with 5-deazaflavin and oxalate. The reduced enzyme did not display an EPR spectrum typical of [4Fe-4S] ferredoxins in the +1 state, unless it was prepared under denaturing conditions. M?ssbauer spectroscopy of reduced 57Fe-enriched amidotransferase confirmed that the cluster was in the +1 state, but the magnetic properties of the reduced cluster observed at 4.2 K indicated that it is characterized by a ground state spin S greater than or equal to 3/2. The midpoint potential of the +1/+2 couple was too low to measure accurately by conventional techniques, but it was below -600 mV, which is 100 mV more negative than reported for [4Fe-4S] clusters in bacterial ferredoxins. Fully reduced amidotransferase had about 40% of the activity of the native enzyme in glutamine-dependent phosphoribosylamine formation. The fact that both the +1 and +2 forms of the enzyme are active indicates that the cluster does not function as a site of reversible electron transfer during catalysis.     

Determination of the nature of the heme environment in nitrosyl indoleamine 2,3-dioxygenase using Multiple-scattering analyses of X-ray absorption fine structure

Multiple-scattering analysis of X-ray absorption fine structure data on the NO adducts of indoleamine 2,3-dioxygenase (IDO) and analysis of X-ray absorption near-edge structure (XANES) have provided the first direct structural information about the iron center for this ubiquitous mammalian metalloprotein. The IDO(II)NO adduct, which is likely to play a physiological role in the immune system, differs from similar adducts such as Mb(II)NO and Lb(II)NO in that the Fe-His bond is essentially broken. At 10 K, the Fe-N(p)(av) bond length = 2.00(2) A, Fe-NO bond length = 1.75 A, and angle = 140 degrees, which are typical of five-coordinate Fe(II)NO species. The XANES is also closer to that of five-coordinate model complexes than six-coordinate species. In addition to the Fe(II)NO species, there was a minor component of the Fe(III)NO adduct because of incomplete reduction of the Fe(II) species. This was also a five-coordinate center and consists of a linear Fe(II)NO(+) moiety with the Fe-N(p)(av) bond length = 2.00(2) A, Fe-NO bond length = 1.63(3) A, and angle = 179 degrees. The results indicate that both the blocking of the heme site to O(2) binding and conformational changes induced by breaking the Fe-N(epsilon) bond may be important mechanisms by which NO inhibits IDO in vitro and in vivo.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. General characteristics and control of electron flow by delta micro H+

A study is presented of the characteristics of redox-linked proton translocation in the b-c1 complex isolated from beef-heart mitochondria and reconstituted into phospholipid vesicles. Measurements of the H+/e- stoichiometry, with three different methods, show that four protons are released from the vesicles per 2e- flowing from quinols to cytochrome c, two of these protons formally deriving from scalar oxidation of quinols by cytochrome c. This H+/e- stoicheiometry is independent of the initial redox state of the b-c1 complex (fully reduced or oxidized) and the rate of electron flow through the complex. It does not change in the pH range 6.0 - 7.2, but declines to 1.5 going with pH from 7.2 - 8.3. This decrease is accompanied by enhancement of the rate of electron flow in the coupled state. Collapse of delta psi effected by valinomycin addition to turning-over b-c1 vesicles resulted in substantial oxidation of cytochrome b-566 and comparable reduction of cytochrome c1, with little oxidation of cytochrome b-562. Nigericin alone had no effect on the steady-state redox levels of b and c cytochromes. Its addition in the presence of valinomycin caused oxidation of b cytochromes but no change in the redox state of cytochrome c1. Valinomycin alone caused a marked enhancement of the rate of electron flow through the complex. Nigericin alone was ineffective, but caused further stimulation of electron flow when added in the presence of valinomycin. The data presented are discussed in terms of two mechanisms: the Q cycle and a model based on combination of protonmotive catalysis by special bound quinone and proton conduction along pathways in the apoproteins.     

The crystal structure of lignin peroxidase at 1.70 A resolution reveals a hydroxy group on the cbeta of tryptophan 171: a novel radical site formed during the redox cycle

The crystal structure of lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium was refined to an R-factor of 16.2 % utilizing synchrotron data in the resolution range from 10 to 1.7 A. The final model comprises all 343 amino acid residues, 370 water molecules, the heme, four carbohydrates, and two calcium ions. Lignin peroxidase shows the typical peroxidase fold and the heme has a close environment as found in other peroxidases. During refinement of the LiP model an unprecedented modification of an amino acid was recognized. The surface residue tryptophan 171 in LiP is stereospecifically hydroxylated at the Cbeta atom due to an autocatalytic process. We propose that during the catalytic cycle of LiP a transient radical at Trp171 occurs that is different from those previously assumed for this type of peroxidase. Recently, the existence of a second substrate-binding site centered at Trp171 has been reported, by us which is different from the "classical heme edge" site found in other peroxidases. Here, we report evidence for a radical formation at Trp171 using spin trapping, which supports the concept of Trp171 being a redox active amino acid and being involved in the oxidation of veratryl alcohol. On the basis of our current model, an electron pathway from Trp171 to the heme is envisaged, relevant for the oxidation of veratryl alcohol and possibly lignin. Beside the opening leading to the heme edge, which can accommodate small aromatic substrate molecules, a smaller channel giving access to the distal heme pocket was identified that is large enough for molecules such as hydrogen peroxide. Furthermore, it was found that in LiP the bond between the heme iron and the Nepsilon2 atom of the proximal histidine residue is significantly longer than in cytochrome c peroxidase (CcP). The weaker Fe-N bond in LiP renders the heme more electron deficient and destabilizes high oxidation states, which could explain the higher redox potential of LiP as compared to CcP.     

The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature.

The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.     

Diffusion-potential-induced oxidation and reduction of cytochromes in chromatophores from Rhodopseudomonas sphaeroides.

A membrane potential jump was induced by the addition of valinomycin in the presence of a KCl concentration gradient across the membrane of Rhodopseudomonas sphaeroides chromatophores. As well as a carotenoid band shift, which is known to be an indicator of membrane potential, absorbance changes due to the oxidation-reduction reactions of cytochromes accompanied the jump. Under aerobic conditions with no reductant added, a part of cytochrome c2 was reduced by an inside-positive potential jump of about 100 mV in the time range of tens of seconds. This can be explained by the location of the cytochrome on the inner side of the chromatophore membrane and electrophoretic flow of electrons across the membrane. On the other hand, in the presence of 1 mM ascorbate, a similar jump of membrane potential induced a rapid oxidation of cytochrome c2 and a subsequent reduction. A rapid reduction of b-type cytochrome was also observed. Antimycin A inhibited the c2 oxidation, but did not inhibit the b reduction. The oxidation of cytochrome c2 may be explained by a diffusion-potential-induced electron flow to cytochrome b and a simultaneous electron donation by cytochrome b and cytochrome c2 to a common electron acceptor, possibly a quinone.     

pH effects on the haem iron co-ordination state in the nitric oxide and deoxy derivatives of ferrous horseradish peroxidase and cytochrome c peroxidase.

The spectral (e.p.r. and absorbance) properties of the NO and deoxy derivatives of ferrous horseradish peroxidase (HRP; EC 1.11.1.7) and baker's-yeast cytochrome c peroxidase (CCP; EC 1.11.1.5) were investigated between pH 7 and pH 2; over the same pH range the kinetics for CO binding were also determined. At neutral pH the e.p.r. and absorption spectra of the NO and deoxy derivatives of HRP and CCP are typical of systems in which the haem iron is in the hexaco-ordinated state and the pentaco-ordinated state respectively. By lowering pH, the e.p.r. and absorption spectra of HRP and CCP undergo reversible transitions, with pKa values of 4.1 for the NO derivatives and less than or equal to 3 for the deoxy derivatives of the ferrous forms. By analogy with O2-carrying proteins and haem model compounds, the pH-dependent spectral changes of HRP and CCP were interpreted as indicative of the protonation of the N(epsilon) atom of the proximal histidine residue and of the cleavage of the Fe-N(epsilon) bond. However, the slow second-order rate constant (0.003 microM-1.s-1) for CO binding to deoxy ferrous HRP and CCP does not increase substantially even at pH 2.6, suggesting that changes in the Fe-haem plane geometry, presumably associated with the cleavage of the Fe-N(epsilon) bond, do not affect appreciably the observed ligand association rate constant.     

Effect of dehydration on electron transport from membrane-bound cytochromes c to bacteriochlorophyll of the photosynthetic reaction center in chromatophores of purple thiobacteria

A method is proposed for spectroscopic probing photo-induced reversible oxidation-reduction changes of high-potential cytochrome in chromatophore films of various humidity. On these preparations of Ect. shaposhnikovii and Chr. minutissium it was found that the characteristic time of cytochrome oxidation, tau, in samples with a high degree of humidity (P/Ps = 0.75) is 2-3 mus, which is close to that seen under physiological conditions (a suspension of intact cells or chromatophores). It was found that under continuous or pulsed illumination the lowering of the relative humidity from 0.75 to 0.15 P/Ps results in a reversible decrease in the amount of cytochrome molecules that can undergo photooxidation. The fraction of cytochrome pool that retains its activity shows a rate of oxidation which remains almost without change. The observed hydration effect and its involvement in the control of the photo-induced oxidation of cytochromes must be taken into account when a model of the molecular mechanism of this process is constructed on the basis of the electron tunneling theory. It is also shown that the dark-reduction kinetics of high-potential cytochrome consist of two components: a fast component with t1/2 = 1-3s which is independent of the sample humidity and a slow component with t1/2 = 5-20 s whose lifetime increases by a factor of 3-5 on reducing the humidity. At a high degree of humidity (P/Ps = 0.75-0.5), the kinetics of cytochrome dark-reduction exhibits only the slow component. The fast component is probably associated with the return of an electron from the primary ferroquinone acceptor and the slow component seems likely to be due to the direct transfer of an electron from exogenous electron donor to the cytochrome.     

Structures of thiolate- and carboxylate-ligated ferric H93G myoglobin: models for cytochrome P450 and for oxyanion-bound heme proteins

Crystal structures of the ferric H93G myoglobin (Mb) cavity mutant containing either an anionic proximal thiolate sulfur donor or a carboxylate oxygen donor ligand are reported at 1.7 and 1.4 A resolution, respectively. The crystal structure and magnetic circular dichroism spectra of the H93G Mb beta-mercaptoethanol (BME) thiolate adduct reveal a high-spin, five-coordinate complex. Furthermore, the bound BME appears to have an intramolecular hydrogen bond involving the alcohol proton and the ligated thiolate sulfur, mimicking one of the three proximal N-H...S hydrogen bonds in cytochrome P450. The Fe is displaced from the porphyrin plane by 0.5 A and forms a 2.41 A Fe-S bond. The Fe(3+)-S-C angle is 111 degrees , indicative of a covalent Fe-S bond with sp(3)-hybridized sulfur. Therefore, the H93G Mb.BME complex provides an excellent protein-derived structural model for high-spin ferric P450. In particular, the Fe-S bond in high-spin ferric P450-CAM has essentially the same geometry despite the constraints imposed by covalent linkage of the cysteine to the protein backbone. This suggests that evolution led to the geometric optimization of the proximal Fe-S(cysteinate) bond in P450. The crystal structure and spectral properties of the H93G Mb acetate adduct reveal a high-spin, six-coordinate complex with proximal acetate and distal water axial ligands. The distal His-64 forms a hydrogen bond with the bound water. The Fe-acetate bonding geometry is inconsistent with an electron pair along the Fe-O bond as the Fe-O-C angle is 152 degrees and the Fe is far from the plane of the acetate. Thus, the Fe-O bonding is ionic. The H93G Mb cavity mutant has already been shown to be a versatile model system for the study of ligand binding to heme proteins; this investigation affords the first structural evidence that nonimidazole exogenous ligands bind in the proximal ligation site.     

Site-directed mutagenesis of tetraheme cytochrome c3. Modification of oxidoreduction potentials after heme axial ligand replacement.

The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.