Zinc-substituted Cytochrome Research Articles

Photoinduced charge separation in single-walled carbon nanotube/protein integrated systems.

Zinc-substituted cytochrome c (Zn-cyt c) is noncovalently bound to single-walled carbon nanotubes (SWNTs), causing the Zn-cyt c fluorescence to be quenched by up to 95%, primarily due to photoinduced charge transfer. Deposition of Zn-cyt c/SWNT films onto conductive oxides allows for harvesting of photoexcited electrons with an internal quantum efficiency of over 5%.

Cathodic photocurrent generation from zinc-substituted cytochrome b562 assemblies immobilized on an apocytochrome b562-modified gold electrode

Hierarchical assemblies of the noteworthy photoactive cytochrome b562 reconstituted with zinc protoporphyrin IX covalently linked with the protein surface were constructed on a gold electrode modified with an apoprotein of cytochrome b562. The integrated photoactive hemoproteins were characterized by electrochemical impedance and quartz crystal microbalance analyses. The protein-immobilized electrode exhibits enhanced photocurrent generation relative to the one having a Zn-substituted hemoprotein monolayer.

Zinc-Substituted Cytochrome P450cam: Characterization of Protein Conformers F420 and F450 by Photoinduced Electron Transfer

Metal substitution of heme proteins is widely applied in the study of biologically relevant electron transfer (ET) reactions. It has been shown that many modified proteins remain in their native conformation and can provide useful insights into the molecular mechanism of electron transfer between the native protein and its substrates. We investigated ET reactions between zinc-substituted cytochrome P450(cam) and small organic compounds such as quinones and ferrocene, which are capable of accessing the protein's hydrophobic channel and binding close to the active site, like its native substrate, camphor. Following the substitution method developed by Gunsalus and co-workers [Wagner, G. C., et al. (1981) J. Biol. Chem. 256, 6262-6265], we have identified two dominant forms of the zinc-substituted protein, F450 and F420, that exhibit different photophysical and photochemical properties. The ET behavior of F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and place themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that F420 corresponds to the open structure of the native cytochrome P450(cam) while F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450(cam). The existence of two distinct conformers of Zn-bound P450(cam) is consistent with the findings of Goodin and co-workers [Lee, Y.-T., et al. (2010) Biochemistry 49, 3412-3419] and has significant consequences for future electron transfer studies on this popular metalloenzyme.

Protein Photoconductors and Photodiodes

Photo finish: Zinc-substituted cytochrome b562 (Zn-cyt b562) immobilized on a gold electrode is an n-type photodiode, whereas zinc-cytochrome c (Zn-cyt c) is a p-type photoconductor (see picture). DFT calculations indicate that the cytochrome band gaps are much lower than those estimated for smaller polypeptides. The semiconductor properties of these proteins depend on the charge distribution on their molecular surfaces.

Zinc porphyrin: A fluorescent acceptor in studies of Zn-cytochromecunfolding by fluorescence resonance energy transfer

FRET between the zinc porphyrin (ZnP) chromophore in zinc-substituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.

Mechanism of Intramolecular Electron Transfer in the Photoexcited Zn-Substituted Cytochrome c: Theoretical and Experimental Perspective

Photoinduced electron transfer (ET) in zinc-substituted cytochrome c (Zn-cyt c) has been utilized in many studies on the long-range ET in protein. Attempting to understand its ET mechanism in terms of electronic structure of the molecule, we have calculated an all-electron wave function for the ground-state of Zn-cyt c on the basis of density functional theory (DFT). The four molecular orbitals (MOs) responsible for excitation by UV-vis light (Gouterman's 4-orbitals) are assigned on the basis of the excited states of chromophore model for Zn-porphine complex calculated with the time-dependent DFT method. ET rates between each Gouterman's 4-orbitals and other MOs were estimated using Fermi's golden rule. It appeared that the two occupied MOs of the 4-orbitals show exclusively higher ET rate from/to particular MOs that localize on outermost amino acid residues (Lys 7 or Asn 54), respectively, whereas ET rates involving the two unoccupied MOs of the 4-orbitals are much slower. These results imply that the intramolecular ET in photoexcited Zn-cyt c is governed by the hole transfer through occupied MOs. The couplings of MOs between zinc porphyrin core and specific amino acid residues on the protein surface have been demonstrated in Zn-cyt c immobilized on an Au electrode via carboxylic acid group-terminated self-assembled monolayer. The Zn-cyt c-modified electrode showed photocurrents responsible for photoillumination. The action spectrum of the photocurrent was identical with the absorption spectrum of Zn-cyt c, indicating photoinduced electron conduction via occupied MOs. The voltage dependence of the photocurrent appeared to be linear and bidirectional like a photoconductor, which strongly supports the intramolecular ET mechanism in Zn-cyt c proposed on the basis of the theoretical calculations.

Triplet State Photosensitization of Nanocrystalline Metal Oxide Electrodes by Zinc-Substituted Cytochrome c: Application to Hydrogen Evolution

The interfacing of nanostructured semiconductor photoelectrodes with redox proteins is an innovative approach to the development of artificial photosynthetic systems. In this paper, we have investigated the photoinduced electron-transfer reactions of zinc-substituted cytochrome c, ZnCyt-c, immobilized on mesoporous, nanocrystalline metal oxide electrodes. Efficient electron injection from the triplet state of ZnCyt-c is observed into TiO(2) electrodes (t(50%) approximately 100 micros) resulting in a long-lived charge-separated state (lifetime of up to 0.4 s). Further studies were undertaken as a function of electrolyte pH and metal oxide employed. Optimum yield of a long-lived charge-separated state was observed employing TiO(2) electrodes at pH 5, consistent with our previous studies of analogous dye-sensitized metal oxide electrodes. The addition of EDTA as a sacrificial electron donor to the electrolyte resulted in efficient photogeneration of molecular hydrogen, with a quantum yield per one absorbed photon of 10 +/- 5%.

Structural analysis of zinc-substituted cytochrome c.

Zinc-substituted cytochrome c has been widely used in studies of protein-protein interactions and photo-induced electron transfer reactions between proteins. However, the coordination geometry of zinc in zinc-substituted cyt c has not yet been determined; two different opinions about the coordination have been reached. Here the solution structures of zinc-substituted cytochrome c that might be five-coordinated and six-coordinated have been refined separately by using (1)H NMR spectroscopy, and the zinc coordination geometry was determined just by NOE distance constraints. Structural analysis of the energy-minimized average solution structures of both the pentacoordinated and hexacoordinated geometries indicate that that zinc in zinc-substituted cyt c should be bound to both His18 and Met80, which means that the zinc is six-coordinated. RMSD values of the family of 25 six-coordinated structures from the average structure are 0.66+/-0.13 A and 1.09+/-0.16 A for the backbone and all heavy atoms, respectively. A statistical analysis of the structure indicates its satisfactory quality. Comparison of the solution structure of the six-coordinated energy-minimized average structure of zinc-substituted cytochrome c with the solution structure of reduced cytochrome c reveals that for the overall folding the secondary structure elements are very close. The availability of the structure provides for a better understanding of the protein-protein complex and for electron transfer processes between Zn cyt c and other metalloproteins.

Mimicking biological electron transport in sol-gel glass: photoinduced electron transfer from zinc cytochrome C to plastocyanin or cytochrome C mediated by mobile inorganic complexes.

Biomimetic studies of electron-transport chains are important for establishing the molecular mechanisms of long-range communications between proteins. We mimic these biological assemblies by encapsulating metalloproteins in sol-gel silica glass and letting mobile inorganic complexes shuttle electrons between the immobilized proteins. We present two examples of such rudimentary electron-transport chains. In both of them the immobilized electron donor is the zinc-substituted cytochrome c, Zncyt; the immobilized electron acceptor is either cupriplastocyanin, pc(II), or ferricytochrome c, cyt(III); and the mobile charge carrier Q/Q(-) is the redox couple FeEDTA(-)(/2)(-) or Ru(NH(3))(6)(3+/2+). The redox processes are photoinduced: Zncyt is excited by the laser pulse and converted to the triplet state, (3)Zncyt, which is a strong reducing agent. Visible absorption, circular dichroism, and electron paramagnetic resonance spectra of the metalloproteins show that encapsulation in sol-gel glass does not affect their intrinsic redox properties. The rigid silica glass spatially separates the proteins from each other. In this matrix, the electron-transfer reactions between (3)Zncyt and pc(II) and between (3)Zncyt and cyt(III), which occur fast in solution, are completely suppressed in the absence of a charge carrier Q/Q(-). The reactivity of FeEDTA(-) and Ru(NH(3))(6)(3+) (as quenchers Q of (3)Zncyt) is minimally affected by the interior of the sol-gel glass. In the glass, the second-order rate constants for the excited-state electron transfer, from (3)Zncyt to Q, are (8.9 +/- 0.6) x 10(6) and (8.0 +/- 2.4) x 10(6) M(-)(1) s(-)(1) for FeEDTA(-) and Ru(NH(3))(6)(3+), respectively. This reaction is followed by the ground-state back electron transfer, from Q(-) to Zncyt(+). In the "monoprotein" glasses Zncyt/Q, the respective second-order rate constants for this back electron-transfer reaction are (4.9 +/- 0.2) x 10(7) and (7.8 +/- 2.7) x 10(7) M(-)(1) s(-)(1). In the "diprotein" glasses Zncyt/Q/pc(II) and Zncyt/Q/cyt(III), containing also the acceptor protein pc(II) or cyt(III), Zncyt(+) decays on two time scales. The faster and major component of this decay is analogous to the only mode of the decay in the Zncyt/Q glasses and is a second-order process. Between 25 and 40% of the initially formed Zncyt(+), however, lives longer (k(slow) =1.1 +/- 0.2 s(-)(1)) and decays by a first-order process. We attribute the lengthening of the Zncyt(+) lifetime to a partial escape of the photogenerated Q(-) into the glass pores, where it reacts with the immobilized pc(II) or cyt(III). Indeed, the visible absorption spectra show the photoinduced reduction of pc(II) and cyt(III). Evidently, the small inorganic complexes, FeEDTA(-)(/2)(-) and Ru(NH(3))(6)(3+/2+), move through the glass pores, react with the encapsulated metalloproteins, and establish the interprotein electron transfer. Each interprotein reaction now occurs in two steps: a mobile charge carrier Q receives an electron from (3)Zncyt, and Q(-) then delivers an electron to pc(II) or cyt(III). Ultimately, the energy of visible light is converted to reducing equivalents for plastocyanin and cytochrome c. The sequential electron transfer described here resembles the events in a rudimentary electron-transport chain. Our findings demonstrate the promise of integrating proteins, with their optimally adjusted redox sites, in photocatalytic materials.

Photoelectrochemical study of Zn cytochrome-c immobilised on a nanoporous metal oxide electrode.

Transient optical spectroscopies and photocurrent action spectra are used to demonstrate photoinduced charge separation between zinc-substituted cytochrome c and a nanocrystalline TiO2 electrode.

Electron transfer reactions in Zn-substituted cytochrome P450cam.

We have investigated photoinduced electron transfer (ET) reactions between zinc-substituted cytochrome P450cam (ZnP450) and several inorganic reagents by using the laser flash photolysis method, to reveal roles of the electrostatic interactions in the regulation of the ET reactions. The laser pulse irradiation to ZnP450 yielded a strong reductant, the triplet excited state of ZnP450, (3)ZnP450, which was able to transfer one electron to anionic redox partners, OsCl(6)(2-) and Fe(CN)(6)(3-), with formation of the porphyrin pi-cation radical, ZnP450(+). In contrast, the ET reactions from (3)ZnP450 to cationic redox partners, such as Ru(NH(3))(6)(3+) and Co(phen)(3)(3+), were not observed even in the presence of 100-fold excess of the oxidant. One of the possible interpretations for the preferential ET to the anionic redox partner is that the cationic patch on the P450cam surface, a putative interaction site for the anionic reagents, is located near the heme (less than 10 A from the heme edge), while the anionic surface is far from the heme moiety (more than 16 A from the heme edge), which would yield 8000-fold faster ET rates through the cationic patch. The ET rate through the anionic patch to the cationic partner would be substantially slower than that of the phosphorescence process in (3)ZnP450, resulting in no ET reactions to the cationic reagents. These results demonstrate that the asymmetrical charge distribution on the protein surface is critical for the ET reaction in P450cam.

Characterization of zinc-substituted cytochrome c by circular dichroism and resonance Raman spectroscopic methods

Iron(III) in cytochrome c is replaced with zinc(II) by a modification of a method published by others, and the procedure is described in full detail. Three forms of cytochrome c—those containing iron(III), iron(II), and zinc(II)—are examined by circular dichroism spectroscopy and resonance Raman spectroscopy. Spectra of both kinds show that introduction of zinc(II) ions does not appreciably alter the overall structure and conformation of cytochrome c. Resonance Raman spectra indicate the size of the porphyrin “core” that is inconsistent with six-coordination and consistent with five-coordination. Unlike the iron(III) and iron(II) ions, which are bound to two axial ligands (His 18 and Met 80), the zinc(II) ion in cytochrome c seems to be bound to only one, most probably His 18. Evidence pertaining to the question of axial coordination is discussed.

Interaction of cytochrome c with flavocytochrome b2.

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 [per cytochrome c reduced; Miles, C. S., Rouviere, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) muM-1 s-1 in the presence of 10 mM L-lactate. This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-transfer rate in the pre-formed complex being in excess of 1000 s-1. Inhibition of the pre-steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate. The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S. & Cambillau, C. (1993) Proteins 16, 408-422] has been tested using site-directed mutagenesis. Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere. The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency. In the steady-state, the turnover rate is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue [Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6345-6350].

Structure of Zinc-Substituted Cytochrome c: Nuclear Magnetic Resonance and Optical Spectroscopic Studies

Optical and proton nuclear magnetic resonance (NMR) studies were carried out to assess the structure of the polypeptide chain and metal ligation in zinc-substituted horse heart cytochrome c (Zn Cyt c). The 1D- and 2D-NMR (COSY, TOCSY, and NOESY) spectra allowed the assignment of proton resonances in 67 amino acid residues. These residues arose from all structural elements of the protein, alpha-helices, beta-turns, and segments of the protein with no defined secondary structure. Small deviations of the chemical shifts of Zn Cyt c proton resonances from native Fe(II) Cyt c of less than 0.1 ppm are due to not fully matching solvent conditions. Differences in the chemical shifts between the two proteins in the range 0.10-0.20 ppm are not clustered and are observed not only in the vicinity of the Zn porphyrin but also on distant surface locations of the cytochrome. The resonance positions of the bridge protons, from the thioether bonds of the porphyrin with Cys 14 and Cys 17, were conserved in Zn Cyt c. Similarly, the Met 80 and His 18 protons had chemical shifts supporting the proposal that His 18 and Met 80, as for Fe(II) Cyt c, may provide the axial ligation in the Zn protein and that zinc may be in an unusual hexacoordinated geometry. Chemical shifts from proton resonances of alternative axial ligands of misfolded cytochrome like His 33, Lys 79, and Phe 82 were found to be the same as in the Fe(II) protein, excluding the possibility of their axial ligation to Zn. The His 18-Zn-Met 80 ligation was also consistent with data from absorption and luminescence studies. We conclude that Zn Cyt c is an adequate structural model for Fe(II) Cyt c as both share the same overall structure, including axial ligands, environment in the porphyrin vicinity, and the same binding interface with redox partners.

Cytochrome c peroxidase binds two molecules of cytochrome c: Evidence for a low-affinity, electron-transfer-active site on cytochrome c peroxidase

We have studied the affinity and stoichiometry of binding of cytochrome c (Cc) to zinc-substituted cytochrome c peroxidase [(ZnP)CcP], which is structurally and electrostatically equivalent to ferrous CcP. Transient absorption spectroscopy has been used to measure both the total quenching of the triplet-state (ZnP)CcP [3(ZnP)CcP] by Fe3+Cc and the fraction of that quenching that is due to electron transfer (et). This redox quenching results in the formation of an intermediate (I) containing the zinc porphyrin pi-cation radical [(ZnP)+CcP] and Fe2+Cc. In titrations of (ZnP)CcP with Fe3+Cc(F) at low ionic strength, where F represents the fungal cytochromes c from Candida krusei, Pichia membranefaciens, or the yeast protein iso-1, the appearance of the et intermediate lags behind the total quenching, with appreciable formation of I occurring only for Cc to CcP ratios > 1. This behavior results from the formation of a 2:1 complex, where one Fe3+Cc(F) binds to a high-affinity domain that exhibits strong quenching yet is et-inactive, while the second Fe3+Cc(F) binds to a low-affinity domain that allows efficient et quenching. At constant concentrations of both proteins, raising the ionic strength eliminates most of the et quenching but reduces the total quenching only minimally, confirming that et occurs preferentially at the low-affinity binding domain, which is the more sensitive to ionic strength. Analogous experiments also favor a 2:1 binding stoichiometry for horse Cc [Cc(horse)] at low ionic strength, with et quenching again proceeding much more favorably in the 2:1 complex than in the 1:1 complex, as with Cc(F). However, the Fe3+Cc(horse) quenches only by electron transfer, unlike the Cc(F). The decay of the triplet-state (ZnP)CcP or magnesium-substituted CcP [(MgP)CcP] was examined during titrations with Fe3+Cc to determine limits for the dissociation rate constant (koff) for the complex. Fe3+Cc(horse) bound to the high-affinity domain in a 1:1 complex at low ionic strength is in rapid exchange, with koff > 50 S-1, whereas Fe3+Cc(F) has koff < 200 s-1. Both types of Fe3+Cc have koff > 10(4)S-1 when they are bound to the low-affinity domain in a 2:1 complex, at both low and high ionic strengths. In contrast, when in the ferrous form, both types of Cc have much lower values of koff (< 10 S-1) at low ionic strength when bound to the low-affinity domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of static and diffusive electron transfer between zinc-substituted cytochrome c and plastocyanin. Indications of nonelectrostatic interactions between highly charged metalloproteins

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetics of static and diffusive electron transfer between zinc-substituted cytochrome c and plastocyanin. Indications of nonelectrostatic interactions between highly charged metalloproteinsJian S. Zhou and Nenad M. KosticCite this: J. Am. Chem. Soc. 1991, 113, 16, 6067–6073Publication Date (Print):July 1, 1991Publication History Published online1 May 2002Published inissue 1 July 1991https://doi.org/10.1021/ja00016a021RIGHTS & PERMISSIONSArticle Views64Altmetric-Citations30LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (846 KB) Get e-Alerts Get e-Alerts

Regulation of interprotein electron transfer by residue 82 of yeast cytochrome c.

Yeast iso-1-cytochrome c (Cc) mutants have been constructed with Phe, Tyr, Gly, Ser, Leu, and Ile at position 82, each with Thr substituted for Cys at position 102. Their long-range electron transfer with zinc-substituted cytochrome c peroxidase (ZnCcP) has been studied by two kinetic techniques. The charge-separated complex, [(ZnCcP)+,FeIICc] converts to [ZnCcP,FeIIICc] by a single, intracomplex electron transfer step that is not governed by "gating" through possible rapid dissociation of the complex or isomerization (for example, heme-ligand) by FeIICc subsequent to its formation from FeIIICc. In every variant with an aliphatic residue at position 82 of Cc, the rate of this electron transfer process is approximately 10(4) slower at approximately 0 degrees C than for the two variants with aromatic residues.

Electron-transfer kinetics of zinc-substituted cytochrome c and its Ru(NH3)5(histidine-33) derivative

The kinetics of the bimolecular electron transfers from triplet-excited zinc-substituted horse heart cytochrome c (Zn-cyt c*) to Ru(NH/sub 3/)/sub 5/L/sup 3 +/ have been measured (L = NH/sub 3/, k(298 K) = 1.4 x 10/sup 7/ M/sup -1/ s/sup -1/; L = histidine, k(298 K) = 2.4 x 10/sup 7/ M/sup -1/ s/sup -1/), along with those of the corresponding thermal back-reactions (L = NH/sub 3/, k(298 K) = 1.5 x 10/sup 8/ M/sup -1/ s/sup -1/; L = histidine, k(298 K) = 3.6 x 10/sup 8/ M/sup -1/ s/sup -1/). The derivatized metalloprotein ruthenium pentaammine histidine-33-zinc-substituted cytochrome c (Ru-Zn-cyt c) has been prepared and characterized by atomic absorption spectroscopy and HPLC analysis of its tryptic digestion fragments. The rate of intraprotein electron transfer from the triplet-excited Zn-porphyrin moiety to the 11.8-A distant Ru(NH/sub 3/)/sub 5/(His-33)/sup 3 +/ residue (k(298 K) = 7.7 x 10/sup 5/ s/sup -1/), as well as that of the thermal back-reaction (k(298 K) = 1.6 x 10/sup 6/ s/sup -1/), has been measured by transient spectroscopy. Intraprotein electron transfer from Ru(II) quenches Zn-cyt c* with a rate constant of 2.4 x 10/sup 2/ s/sup -1/. These kinetics are discussed in terms of the semiclassical theorymore » of electron-transfer reactions.« less

Yeast cytochrome c with phenylalanine or tyrosine at position 87 transfers electrons to (zinc cytochrome c peroxidase)+ at a rate ten thousand times that of the serine-87 or glycine-87 variants.

Of the many factors known to influence the rate of electron transfer between two metalloproteins, it is particularly difficult to assess the role of the polypeptide matrix intervening between the donor and acceptor sites. To determine whether the phylogenetically conserved Phe-87 of yeast iso-1-cytochrome c helps to mediate electron transfer between cytochrome c and cytochrome c peroxidase, we have constructed mutants of cytochrome c that are altered at this position and now have studied the kinetics of long-range electron transfer within their complexes with zinc-substituted cytochrome c peroxidase. We find that the rate of electron transfer from reduced cytochrome c to the zinc cytochrome c peroxidase pi-cation radical is four orders of magnitude greater when phenylalanine or tyrosine is present at position 87 than when serine or glycine is present.

Photoinduced long-range electron transfer in the zinc-substituted cytochrome c/cytochrome c peroxidase complex

Photoinduced long-range electron transfer in the zinc-substituted cytochrome c/cytochrome c peroxidase complex