Biomedical subjects

G Tollin

Publications and source records attributed to G Tollin.

At least 73 records · Page 4Linked to original sources

PubMed1995

Low concentrations of diacylglycerol promote the binding of apolipophorin III to a phospholipid bilayer: a surface plasmon resonance spectroscopy study.

The binding of the exchangeable apolipoprotein apolipophorin III (apoLp-III) to an egg phosphatidylcholine bilayer as a function of the concentration of diacylglycerol (DG) in the bilayer was studied by surface plasmon resonance spectroscopy. At a DG concentration of 2 mol % in the bilayer, the binding of apoLp-III reached saturation. Under saturating conditions, apoLp-III forms a closely packed monolayer approximately 55 A thick, in which each molecule of protein occupies approximately 500 A2 at the membrane surface. These dimensions are consistent with the molecular size of the apoLp-III molecule determined by x-ray crystallography, if apoLp-III binds to the bilayer with the long axis of the apoLp-III normal to the membrane surface. In the absence of protein, the overall structure of the lipid bilayer was not significantly changed up to 2.5 mol% DG. However, at 4 and 6 mol % DG, the presence of nonbilayer structures was observed. The addition of apoLp-III to a membrane containing 6 mol % DG promoted the formation of large lipid-protein complexes. These data support a two-step sequential binding mechanism for binding of apoLp-III to a lipid surface. The first step is a recognition process, consisting of the adsorption of apoLp-III to a nascent hydrophobic defect in the phospholipid bilayer caused by the presence of DG. This recognition process might depend on the presence of a hydrophobic sensor located at one of the ends of the long axis of the apoLp-III molecule but would be consolidated through H-bond and electrostatic interactions. Once primary binding is achieved, subsequent enlargement of the hydrophobic defect in the lipid surface would trigger the unfolding of the apolipoprotein and binding via the amphipathic alpha-helices. This two-step sequential binding mechanism could be a general mechanism for all exchangeable apolipoproteins. A possible physiological role of the ability of apoLp-III to bind to lipid structures in two orientations is also proposed.

Animals↗

PubMed1995

Transient kinetics of intracomplex electron transfer in the human cytochrome b5 reductase-cytochrome b5 system: NAD+ modulates protein-protein binding and electron transfer.

Transient kinetics of reduction and interprotein electron transfer in the human cytochrome b5 reductase-cytochrome b5 (b5R-b5) system was studied by laser flash photolysis in the presence of 5-deazariboflavin and EDTA at pH 7.0. Flash-induced reduction of the FAD cofactor of b5R by deazariboflavin semiquinone (in the absence of b5) occurred in a rapid second-order reaction (k2 = 3.1 x 10(8) M-1 s-1) and resulted in a neutral (blue) FAD semiquinone. The heme of cytochrome b5 (in the absence of b5R) was also rapidly reduced in this system with k2 = 3.1 x 10(8) M-1 s-1. When the two proteins were mixed at low ionic strength, a strong complex was formed. Although the heme of complexed b5 could be directly reduced by deazariboflavin semiquinone, the second-order rate constant was nearly an order of magnitude smaller than that of free b5 (k2 = 3.4 x 10(7) M-1 s-1). In contrast, access to the FAD of b5R by the external reductant was decreased by considerably more than an order of magnitude (k2 < 1 x 10(7) M-1 s-1). When an excess of b5R was titrated with small increments of b5 and then subjected to laser flash photolysis in the presence of deazariboflavin/EDTA, interprotein electron transfer from the b5R FAD semiquinone to the heme of b5 could be observed. At low ionic strength (I = 16 mM), the reaction showed saturation behavior with respect to the b5 concentration, with a limiting first-order rate constant for interprotein electron transfer k1 = 375 s-1, and a dissociation constant for protein-protein transient complex formation of approximately 1 microM. The observed rate constants for interprotein electron transfer decreased 23-fold when the ionic strength was increased to 1 M, indicating a plus-minus electrostatic interaction between the two proteins. Saturation kinetics were also observed at I = 56, 96, and 120 mM, with limiting first-order rate constants of 195, 155, and 63 s-1, respectively. In the presence of NAD+, the transient protein-protein complex was stabilized by approximately a factor of two, and limiting first-order rate constants of 360 s-1 were obtained at both I = 56 mM and I = 96 mM and 235 s-1 at I = 120 mM. Thus, NAD+ appears to stabilize as well as to optimize the protein-protein complex with respect to electron transfer. Another effect of NAD+ is to appreciably slow autoxidation and disproportionation of the FAD semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome Reductases↗

PubMed1995

Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation.

The stability properties of oxidized wild-type (wt) and site-directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287-3295); we also find this to be true with respect to Gdn-HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X-ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗

PubMed1995

Use of flavin photochemistry to probe intraprotein and interprotein electron transfer mechanisms.

Photoexcitation of flavin analogs generates the lowest triplet state (via intersystem crossing from the fist excited singlet state) in the nanosecond time domain and with high quantum efficiency. The triplet, being a strong oxidant, can abstract a hydrogen atom (or an electron) from a reduced donor in a diffusion-controlled reaction. If the donor is a redox protein, the oxidation process can be used to initiate an electron transfer sequence involving either intramolecular or intermolecular reactions. If the donor is an organic compound such as EDTA, the neutral flavin semiquinone will be produced by H atom abstraction; this is a strong reductant and can subsequently transfer a hydrogen atom (or an electron) to an oxidized redox protein, thereby again initiating a sequence of intramolecular or intermolecular processes. If flavin photoexcitation is accomplished using a pulsed light source, the initiation of these protein electron transfer reactions can be made to occur in the nanosecond to microsecond time domain, and the sequence of events can be followed by time-resolved spectrophotometry to obtain rate constants and thus mechanistic information. The present paper describes this technology, and selected examples of its use in the investigation of redox protein mechanisms are given.

Cytochromes↗

PubMed1995

Structure-function relationships in the ferredoxin/ferredoxin: NADP+ reductase system from Anabaena.

We have used a combination of laser flash photolysis time-resolved spectrophotometry and site-specific mutagenesis of surface amino acid residues to investigate the structural factors which influence electron transfer from Anabaena ferredoxin to its physiological partner ferredoxin-NADP+ reductase. Two ferredoxin residues (E94 and F65) are found to be highly critical interaction sites, whereas other nearby residues are found to be either inconsequential or to have only moderate effects. Basic residues near the N-terminus of the reductase are also found to exert a significant influence on interprotein electron transfer. The mechanistic implications of these results are discussed.

Anabaena↗

PubMed1995

Photobleaching of the photoactive yellow protein from Ectothiorhodospira halophila promotes binding to lipid bilayers: evidence from surface plasmon resonance spectroscopy.

The photoactive yellow protein (PYP) from the phototrophic bacterium Ectothiorhodospira halophila is a small, soluble protein that undergoes reversible photobleaching upon blue light irradiation and may function to mediate the negative phototactic response. Based on previous studies of the effects of solvent viscosity and of aliphatic alcohols on PYP photokinetics, we proposed that photobleaching is concomitant with a protein conformational change that exposes a hydrophobic region on the protein surface. In the present investigation, we have used surface plasmon resonance (SPR) spectroscopy to characterize the binding of PYP to lipid bilayers deposited on a thin silver film. SPR spectra demonstrate that the net negatively charged PYP molecule can bind in a saturable manner to electrically neutral, net positively, and net negatively charged bilayers. Illumination with either blue or white light of a PYP solution, which is in contact with the bilayer, at concentrations below saturation results in an increase in the extent of binding, consistent with exposure of a high affinity hydrophobic surface in the photobleached state, a property that may contribute to its biological function. A value for the thickness of the bound PYP layer (23 A), obtained from theoretical fits to the SPR spectra, is consistent with the structure of the protein determined by x-ray crystallography and indicates that the molecule binds with its long axis parallel to the membrane surface.

Adsorption↗

PubMed1994

Conformational changes in rhodopsin probed by surface plasmon resonance spectroscopy.

Surface plasmon resonance (SPR) spectroscopy has been used to follow incorporation and light-induced conformational changes in bovine rhodopsin reconstituted into an egg phosphatidylcholine bilayer deposited on a thin silver film. The magnitude of the SPR spectral changes caused by light varies with pH in a manner paralleling that in flash photolysis experiments, which monitor formation of metarhodopsin II. Irradiation produces an increase of approximately 4 A in the average thickness of the proteolipid layer, consistent with exposure of recognition sites for the G protein. The results demonstrate that the SPR technology described herein may be used to monitor conformational events in membrane-associated receptors such as rhodopsin.

Animals↗

PubMed1994

Intramolecular electron transfer in yeast flavocytochrome b2 upon one-electron photooxidation of the fully reduced enzyme: evidence for redox state control of heme-flavin communication.

Flavocytochrome b2, which has been fully reduced using L-lactate, can be rapidly oxidized by 1 equiv using the laser-generated triplet state of 5-deazariboflavin. Parallel photoinduced oxidation occurs at the reduced heme and at the fully reduced FMN (FMNH2) prosthetic groups of different enzyme monomers, producing the anion semiquinone of FMN and a ferric heme. Following the initial oxidation reaction, rapid intramolecular reduction of the ferric heme occurs with concomitant oxidation of FMNH2, generating the neutral FMN semiquinone. The observed rate constant for this intramolecular electron transfer is 2200 s-1, which is 1 order of magnitude larger than the turnover number under these conditions. A slower reduction of the heme prosthetic group also occurs with an observed rate constant of approximately 10 s-1, perhaps due to intersubunit electron transfer from reduced FMN to heme. The rapid intramolecular electron transfer between the FMNH2 and ferric heme is eliminated upon addition of excess pyruvate (Ki = 3.8 mM). This latter result indicates that pyruvate inhibition of catalytic turnover apparently can occur at the FMNH2-->heme electron transfer step. These results markedly differ from those previously obtained (Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555) and confirmed here for electron transfer within the one-electron reduced enzyme and for the effect of pyruvate binding, suggesting that intramolecular communication between the heme and flavin prosthetic groups can be controlled by the redox state of the enzyme and by ligand binding to the active site.

Electron Transport↗

PubMed1994

Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy.

The structural properties of lipid films, made from a squalene/butanol solution containing varying amounts (0-15 mg/ml) of egg phosphatidylcholine and deposited on a thin metallic silver layer, were investigated using surface plasmon resonance (SPR) spectroscopy. Optical parameters (thickness, refractive index and extinction coefficient) of such supported self-assembled lipid membranes were obtained from a theoretical analysis of the experimental SPR curves. The mass of the lipid membrane and the area and volume occupied by one lipid molecule were also calculated. The results were consistent with the formation of durable and homogeneous lipid bilayers on the solid substrate, and indicated similarities in structural properties between the present lipid bilayers and freely suspended and Langmuir-Blodgett bilayer membranes. Such bilayers represent a simple model for biological membranes, as well as providing a means of immobilizing proteins for various practical applications, including receptor-based sensors and molecular devices. The results confirm the value of the SPR technique for investigating the properties of thin biomolecular dielectric films deposited on a metal surface.

Lipid Bilayers↗

PubMed1994

Direct measurement by laser flash photolysis of intramolecular electron transfer in the three-electron reduced form of ascorbate oxidase from zucchini.

Ascorbate oxidase, which has been fully reduced by its substrate, can rapidly transfer a single electron to the laser-generated triplet state of 5-deazariboflavin. Subsequent to this, intramolecular electron transfer occurs resulting in the oxidation of the blue type I copper center. This latter process proceeds via biphasic kinetics, with observed rate constants of 9500 s-1 and 1400 s-1, both of which are protein concentration independent. This indicates that the initial oxidation reaction involves the type II, III trinuclear center, probably occurring via parallel reactions of two of the three copper atoms. The rate constants for intramolecular electron transfer in the three-electron reduced enzyme are one to two orders of magnitude larger than previously observed for the one-electron reduced enzyme, indicating a dramatic effect of the redox state of the enzyme on the intramolecular communication between the copper centers.

Ascorbate Oxidase↗

PubMed1994

Further characterization by site-directed mutagenesis of the protein-protein interface in the ferredoxin/ferredoxin:NADP+ reductase system from Anabaena: requirement of a negative charge at position 94 in ferredoxin for rapid electron transfer.

Ferredoxins are small electron transfer proteins found ubiquitously in nature. In green plant photosynthesis, the soluble [2Fe-2S] ferredoxin shuttles electrons from Photosystem I to ferredoxin:NADP+ reductase. In order to define the features of the protein/protein interface required for efficient electron transfer from ferredoxin to ferredoxin:NADP+ reductase, we have made site-directed mutants of the ferredoxin from the cyanobacterium Anabaena 7120 and measured the rate constants for electron transfer to ferredoxin:NADP+ reductase using laser flash photolysis. Previous results from this laboratory identified two residues in ferredoxin that were crucial to electron transfer between these proteins. One such position (F65) was subsequently shown to require an aromatic amino acid, and it was further shown that interprotein electron transfer was rate-limiting in the case of the slowly reacting F65A mutant (Hurley et al., 1993 J. Am. Chem. Soc. 115, 11,698-11,701). The second crucial position (E94) is shown in the present study to require a negative charge in order to maintain wild-type-like electron transfer reactivity toward ferredoxin:NADP+ reductase. Further, we also demonstrate, for the slowly reacting E94Q mutant, that electron transfer is the rate-limiting step in the interprotein interaction.

Anabaena↗

PubMed1994

A thermodynamic study by laser-flash photolysis of plastocyanin and cytochrome c6 oxidation by photosystem I from the green alga Monoraphidium braunii.

Plastocyanin and cytochrome c6 from the green alga Monoraphidium braunii reduce the photo-oxidized algal photosystem I (PSI) reaction center chlorophyll (P700) with similar kinetics, as expected from their functional equivalence. The observed P700+ reduction rate constants show a non-linear dependence on metalloprotein concentration, which indicates a (minimal) two-step kinetic mechanism involving complex formation prior to electron transfer. The dependence of the observed rate constants on NaCl concentration suggests that the electrostatic interaction forces between the negatively charged donor proteins and PSI are repulsive at neutral pH and relatively low ionic strength (I), although attractive dipole-dipole interactions may play a role at higher ionic strengths. Activation parameters for P700+ reduction by cytochrome c6 and plastocyanin have been determined by studying the temperature dependence of the respective rate constants at varying ionic strength and pH. Changes in NaCl concentration and pH induce significant changes in the activation free energy of the overall reaction, even though the corresponding values for activation enthalpy and entropy undergo changes in opposite directions. Such a compensation effect between enthalpy and entropy is observed with both cytochrome c6 and plastocyanin. Protein concentration dependencies of the observed rate constants at different temperatures has allowed an estimate of the free energy change during complex association, as well as the activation parameters for electron transfer, according to a two-step kinetic model.

Chlorophyta↗

PubMed1994

Transient kinetic and oxidation-reduction studies of spinach ferredoxin:nitrite oxidoreductase.

The oxidation-reduction midpoint potentials for the two prosthetic groups of the chloroplast-located, ferredoxin-dependent nitrite reductase of spinach leaves have been determined by spectroelectrochemical titrations and cyclic voltammetry. The average of the results obtained by the two techniques are Em = -290 mV for the siroheme group and Em = -365 mV for the [4Fe-4S] cluster. The value obtained for the [4Fe-4S] cluster is substantially more positive than values obtained previously in experiments which utilized electron paramagnetic resonance spectroscopy at cryogenic temperatures to monitor the reduction state of the cluster. Laser flash photolysis experiments have been used to monitor electron transfer from reduced ferredoxin to nitrite reductase and have provided the first evidence for electron transfer between the two prosthetic groups of the enzyme. The effect of ionic strength on the observed kinetics has provided support for the proposal that electrostatic interactions between ferredoxin and nitrite reductase play an important role in the reaction mechanism.

Kinetics↗

PubMed1994

Investigation of the rate limiting step for electron transfer from NADPH:cytochrome P450 reductase to cytochrome b5: a laser flash-photolysis study.

The reduction kinetics of the one-electron-reduced cytochrome P450 reductase:cytochrome b5 complex (P450R1e:b5ox) has been investigated by the laser flash-photolysis technique, using the semiquinone of 5-deazariboflavin (5-dRfH.) as the reductant. Investigation of the kinetic properties of the individual components at 470 nm indicated that P450R1e and b5ox were reduced via second-order kinetics by 5-dRfH. with rate constants of 1 x 10(8) M-1 s-1 and 4.2 x 10(8) M-1 s-1, respectively. Intramolecular electron transfer from (laser reduced) FADH. to FMNH. was measured at 585 nm and a first-order rate constant of 36 s-1 was obtained for this process. Reduction of the preformed P450R1e:b5ox complex by 5-dRfH. was biphasic and second-order rate constants of 5.4 x 10(8) M-1 s-1 and 7.5 x 10(7) M-1 s-1 were obtained for the fast and slow phases of reduction. The time-resolved flash-induced difference spectrum was consistent with the simultaneous direct reduction (by 5-dRfH.) of protein-bound flavin and heme, followed by an additional slower first-order intracomplex electron transfer (kLim = 37 s-1) from protein-bound flavin semiquinone to heme. The results indicate that the (laser-generated) two-electron-reduced form of cytochrome P450 reductase is catalytically competent in the transfer of reducing equivalents to oxidized cytochrome b5 and suggest that formation of FMNH2 as a result of internal electron transfer from FADH. to FMNH. within P450R1e is the rate limiting step in the reduction of cytochrome b5 by cytochrome P450 reductase. The Kd of the cytochrome P450 reductase:cytochrome b5 complex was estimated to be approximately 1.5 x 10(-6) M.

Animals↗

PubMed1994

Comparison of electron transfer kinetics between redox proteins free in solution and electrostatically complexed to a lipid bilayer membrane.

The second-order rate constants obtained in solution for the reduction of horse cytochrome c (cytc; net charge +7) by either Clostridium beijerinckii flavodoxin semiquinone (Fld; net charge -16) or reduced spinach ferredoxin (Fd; net charge -15) decrease monotonically with increasing ionic strength, as expected for reactions between oppositely charged species. Although the rate constant for the Fld reaction is almost two orders of magnitude larger at low ionic strength than that for Fd, the values extrapolated to infinite ionic strength are closely similar, indicating comparable reactivities when electrostatic effects are eliminated. Furthermore, Fld has a much larger value for the electrostatic interaction energy, and thus a larger apparent active site charge, than does Fd, accounting for the rate constant disparity at low ionic strength. Electrostatically binding cytc at low ionic strength to a negatively charged lipid bilayer vesicle (membranes containing mixtures of egg phosphatidylcholine (PC) and cardiolipin (CL)) results in a marked decrease of the observed electron transfer rate constant (k(obs)) for reduction of the cytochrome by both Fld and Fd. The magnitude of this decrease is proportional to the mole percent of CL present in the membrane (10- to 20-fold change over 5-60 mol%). With Fld, k(obs) decreases monotonically with increasing ionic strength at a fixed CL concentration. With Fd an increase in k(obs) occurs as the ionic strength is increased, which maximizes at intermediate ionic strength at a value larger than that obtained in the absence of lipid vesicles. When Fld is electrostatically bound to a positively charged vesicle composed of 40 mol% dioctadecyldimethylammonium ion (DODAC) and 60 mol% PC, again k(obs) for electron transfer to cytc is decreased over that obtained in solution, and the magnitude is diminished monotonically by increasing ionic strength. In contrast, k(obs) for electron transfer from Fd to cytc is unaffected by the presence of the positively charged membrane. The implications of these results for the role of membrane surface charge in modulating protein-protein interactions is discussed.

Cardiolipins↗

PubMed1994

A "parallel plate" electrostatic model for bimolecular rate constants applied to electron transfer proteins.

A "parallel plate" model describing the electrostatic potential energy of protein-protein interactions is presented that provides an analytical representation of the effect of ionic strength on a biomolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site-specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.

Binding Sites↗

PubMed1994

Structure-function studies of [2Fe-2S] ferredoxins.

The ability to overexpress [2Fe-2S] ferredoxins in Escherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegatative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in in these proteins in their oxidized and reduced states has been studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

Amino Acid Sequence↗

PubMed1994

Protein interaction sites obtained via sequence homology. The site of complexation of electron transfer partners of cytochrome c revealed by mapping amino acid substitutions onto three-dimensional protein surfaces.

Amino acid substitutions in all but the most divergent of cytochromes c have been categorized as being conservative or radical and mapped onto the three-dimensional structure of yeast cytochrome c. Color-coded, space-filling representations reveal a large 24 A diameter surface area which is invariant or conservatively substituted on the front left face of the cytochrome c molecule. Chemical modifications and mutations which inhibit complex formation and electron transfer with reaction partners also map to this surface. In sharp contrast, the back side of the protein is randomly substituted with both conservative and radical replacements. The invariant/conservatively substituted surface on the front of cytochrome c thus defines the site of interaction with redox partners and provides a measure of its dimensions. Further, this analysis strongly suggests that there is only a single site of oxidation and reduction on cytochrome c for all of its physiological reactions. The same analysis applied to bacterial cytochrome c2 shows that its conserved surface is similar in size and location to that of cytochrome c. Analyses of native and model reaction partners of cytochromes c and c2, such as cytochrome b5, plastocyanin, and bacterial photosynthetic reaction centers, also reveal probable active site surfaces for complexation and electron transfer, which are complementary in size to that of the c-type cytochromes. The availability of a three-dimensional structure and of several closely related amino acid sequences for a given functional class of protein is the only limitation on this type of analysis, which can then serve as a basis for designing site-directed mutagenesis experiments.

Binding Sites↗