Interaction of cytochromec with phospholipid monolayers and bilayer membranes

Summary For the study of the interaction between oxidized cytochromec and phosphatidylinositide, two different model systems were used: (1) monolayers which were deposited after the method of Langmuir and Blodgett onto glass plates, and (2) bimolecular (“black”) membranes in aqueous phase. The amount of bound protein was determined with a sensitive spectrophotometer. It was found that at low ionic strength about 10¹³ cytochromec molecules per cm² are bound to the lipid surface, which nearly corresponds to a densely packed monolayer. At high ionic strength (∼ 0.1m) or low pH (pH<3), the adsorbed protein layer becomes unstable. This result indicates that the interaction is mainly electrostatic. In accordance with this conclusion is the observation that the rate of adsorption is diffusion controlled; i.e., almost every protein molecule hitting the surface is bound. The cytochromec monolayer can be reduced by ascorbate. In contrast to ferrocytochromec in solution, the bound ferrocytochrome was found to be autoxidable.     

Simultaneous measurements of optical and electrical properties of artificial membranes composed of mitochondrial lipids and their interaction with cytochromec

Summary A newly constructed cell, which allows simultaneous measurements of optical and electrical properties, was used to study bimolecular black membranes composed of beef heart mitochondrial lipids and their interaction with cytochromec.The results show that these highly charged membranes are stable only in relatively limited ranges of boundary conditions. In 0.1n KCl their maximum direct current (dc) resistance is 7×10⁸ Ohm cm²±10%; the series capacity at 1kHz is 0.43 F/cm²±3%; the entire thickness, determined by optical reflectivity, is 5.8±0.2 nm.The interaction between oxidized cytochromec and these lipid membranes is primarily of electrostatic nature, and dependent on the presence of highly charged phospholipids, such as diphosphatidyl glycerol (cardiolipin) and phosphatidyl ethanolamine. The attachment of cytochromec maximally causes a 2.5-fold increase in reflectivity, without any noticeable change in the capacity. This leads to a subsequent instability of the membrane (i.e., rupture) preceded by a rapid increase of the dc conductivity. This behavior is far less pronounced with reduced cytochromec.     

Functional morphology of the pars intermedia of the rat hypophysis as revealed with the electron microscope

Summary The pars intermedia of the hypophysis of normal and experimental rats was studied by electron microscopy. Observations of the hypophysis at various intervals following formalin induced stress or adrenalectomy indicate the existence of a functional relationship between the posterior lobe, the pars intermedia, and the adrenal cortex.Glandular cells of the normal pars intermedia are divided into two types, i. e., the light and dark cells. The former type dominates in number and is characterized by a large amount of cytoplasm filled with clear vesicles 250–350 m in diameter. Dark secretory granules smaller than 300 m are few in number and restricted to the Golgi region.After a single injection of formalin, the clear vesicles of the light cell dimmish and dark secretory granules varying in opacity increase in number. Transition from dark granules to clear vesicles is suggested. Three to five days after adrenalectomy, the light cells contain an abundance of moderately dense vesicles which are smaller than the larger more electron lucent vesicles of the normal light cells. The moderately dense vesicles are about 200 m in diameter and are extremely abundant filling the entire cytoplasm of the light cells 7 days after adrenalectomy.Bundles of unmyelinated nerve fibers are often observed in the pars intermedia, and a typical neuroglandular synapse was found in the pars intermedia of a sham-operated animal suggesting neural control of the secretion process of pars intermedia cells.The author wishes to express his hearty thanks to Dr. K. Kurosumi for his guidance throughout this work.     

Phosphorylation of heavy sarcoplasmic reticulum vesicles: Identification and characterization of three phosphorylated proteins

Summary Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl₂, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 m -³²P-ATP.³²P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl₂ (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 m sodium dantrolene, an inhibitor of Ca²⁺ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.     

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK _m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK _d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK _i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK _d 1.05±0.13nM and apparentK _i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [³H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM _r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.     

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Kinetic studies of the lipid requirement of mitochondrial cytochromec oxidase

The lipid requirement of cytochromec oxidase was reinvestigated using both acetone and phospholipase A to deplete mitochondria of lipid. Removal of lipid resulted in a decrease in both the apparentK _m for cytochromec and apparentV _max when compared to control mitochondria. Addition of phospholipid to the assay mixture reactivated the enzyme. For both treatments theK _m returned to the control value. With phospholipase A treated mitochondria theV _max increased to near the control value, while acetone extracted mitochondria could be restored to aV _max of 1/2 that of the control. Detergent does not substitute for phospholipid and inhibits the reactivation with phospholipid.This research was supported in part by United States Public Health Service Research Grant AM-14632 and a Grant-in-Aid of the American Heart Association.     

Interactions involving the cyanine dye,diS-C3-(5), cytochromec and liposomes and their implications for estimations of Δψ in cytochromec oxidase-reconstituted proteoliposomes

Summary The interference of cytochromec with absorption and fluorescence changes of the cyanine dye, diS-C₃-(5), was investigated in the presence of liposomes and cytochromec-oxidase reconstituted proteoliposomes. The apparent cytochromec-dependent quenching of diS-C₃-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochromec-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochromec-induced breakdown of dye was observed in the presence of hydrogen peroxide.The cytochromec mediated absorbance and fluorescence losses of diS-C₃-(5) in liposomal or proteoliposomal systems are prevented by Ca²⁺ and La³⁺ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochromec is inhibited either directly (e. g. by BHT) or indirectly, by preventing the binding of cytochromec to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential inaa ₃-reconstituted proteoliposomes is discussed.     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

Structure and evolution of cytochrome oxidase

The structural features of cytochrome oxidases are reviewed in light of their evolution. The substrate specificity (quinol vs. cytochromec) is reflected in the presence of a unique copper centre (Cu _A ) in cytochromec oxidases. In several lines of evolution, quinol oxidases have independently lost this copper. Also, the most primitive cytochromec oxidases do not contain this copper, and electron entry takes place viac-type haems. These enzymes, exemplified by the rhizobial FixN complex, probably remind the first oxidases. They are related to the denitrification enzyme nitric oxide reductase.     

A model mosaic membrane: Association of phospholipids and cytochrome oxidase

The structure and physical properties of model membranes formed from lipids and cytochromec oxidase have been examined. The lipid-depleted protein is in the form of 90 Å rods or globules. When phospholipid is added the rods swell and then. form sheets and concentric membrane vesicles. The protein is saturated with lipid at 65 g/atoms of phosphorus per mole of hemea. Electron microscope examination by negative staining, sectioning, and freeze etching indicates a 50 Å thick unit membrane with 50–60 Å protein globules in the lipid bilayer. Infrared, circular dichroism and fluorescence binding studies are consistent with globular protein units surrounded with lipid. Diolein will substitute for phospholipid but the membrane formed remains as sheets rather than vesicles. Saturated phospholipids will not interact with the oxidase to form membrane. The capacity to form membrane is specific to protein associated with the hemea, and other insoluble protein in the original oxidase preparation cannot form membrane.     

The ultrastructural basis for the identification of cell types in the pancreatic islets I. Guinea pig

Summary The pancreatic islets of the guinea pig have been studied by light and electron microscopy. The B granules in glutaraldehyde-fixed tissue often are cup-shaped with an indentation visible on one side of the granule. Phosphotungstic acid hematoxylin (PTAH) positive cells have been characterized by electron microscopy as three subtypes based on the size of the secretory granules. A_a cells are the most common and have secretory granules around 200 m in diameter. A_b cells have large secretory granules around 300 m in diameter and are relatively infrequent. A_c cells are the least common and have small (160 m) granules. Characteristic D cells are identifiable by electron microscopy and, on the basis of the subsequent study (Munger, Caramia, and Lacy, 1965), are identified as the silver positive cells observed by light microscopy.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases.-The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donohue.     

A Ca-dependent K channel in “luminal” membranes from the renal outer medulla

Summary This paper describes properties of⁸⁶Rb fluxes through K channels in luminal membrane vesicles prepared from rabbit renal outer medulla. By measuring⁸⁶Rb uptake against an opposing chemical gradient of K ions, using membranes loaded with KCl, a transient accumulation of isotope is observed, which is blocked by Ba ions. This is the behavior expected of a conductive Rb flux through a Ba-sensitive K channel. The⁸⁶Rb accumulation is driven by an electrical diffusion potential as shown in experiments using either vesicles loaded with different anions, or an outwardly directed Li gradient with a Li ionophore. The vesicles containing the channel show a cation selectivity with the order Rb > K > Cs > Li > Na > choline. The Ba-sensitive Rb flux is dependent on Ca within the vesicles, with a very high affinity estimated asK _0.5 10 to 100nm. The vesicles appear to be right-side-out. The Ba-sensitive⁸⁶Rb uptake is also inhibited by quinineK _0.5 30 m but is insensitive to tetraethyl ammonium ions and apamin. These isotope flux experiments complement electrophysiological experiments in providing independent evidence for the existence of K channels in the luminal surface of cells of this ascending limb of the loop of Henle. The very high Ca affinity suggests that cytoplasmic Ca could play an important role in regulation of transepithelial salt flux in this region of the nephron.     

Calcium at the surface of cardiac plasma membrane vesicles: Cation binding,surface charge screening,and Na−Ca exchange

Summary Calcium binding and Na–Ca exchange activity were measured in isolated cardiac plasma membrane vesicles under various ionic conditions. A model was developed to describe the Ca binding characteristics of cardiac sarcolemmal vesicles using the Gouy-Chapman theory of the diffuse double layer with specific cation binding to phospholipid carboxyl and phosphate groups. The surface association constants used for Ca, Na, K and H binding to both of these groups were 7, 0.63, 0.3 and 3800m ^–1, respectively. This model allows the estimation of surface [Ca] under any specific ionic conditions. The effects of the divalent screening cation, dimethonium, on Ca binding and Na–Ca exchange were compared. Dimethonium had no significant effect on Ca binding at high ionic strength (150mm KCl), but strongly depressed Ca binding at low ionic strength. Dimethonium had no significant effect on Na–Ca exchange (Na-inside dependent Ca influx) at either high or low ionic strength. These results suggest that the Ca sites of the Na–Ca exchanger are in a physical environment where they are either not exposed to or not sensitive to surface [Ca].     

Synapses in the cerebral ganglion of adult Ciona intestinalis

Summary Electron microscopy of the synaptic morphology of synapses in the cerebral ganglion of the adult ascidian (sea squirt) Ciona intestinalis reveals that the synapses are restricted to the central neuropil of the ganglion. Many of the synapses show a polarity of structure such that pre and post synaptic parts can be identified. The vesicles in the presynaptic bag are of two main diameters 80 and 30 nm respectively. The large vesicles have electron dense contents that vary both in their capacity and dimensions.The pre and postsynaptic membranes are more electron dense than the surrounding membranes, but they are only slightly thicker. Both the pre and post synaptic membranes have electron dense dots some 10 nm in diameter associated with their cytoplasmic surfaces. Sometimes the presynaptic membrane has larger peg-like projections between the vesicles. Associated with the post synaptic membrane are tubules some 10 nm in diameter. These tubules may be the dots cut obliquely.The synaptic cleft material is more electron dense than the surrounding intercellular material, and in it there is a dense line made up of granules about 3–5 nm in diameter. This dense line is usually mid way between the pre and post synaptic membranes, but may be nearer the postsynaptic membrane.No tight junctions between adjacent nerve process profiles have been observed.I wish to thank Professors J. Z. Young, F. R. S. and E. G. Gray for much advice and encouragement, also Dr. R. Bellairs for the use of electron microscope facilities and Mr. R. Moss and Mrs. J. Hamilton for skillful technical assistance.     

Differential exposure of components of cytochromeb−c 1 region in beef heart mitochondria and electron transport particles

The reduction of cyctochromesc +c ₁ by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochromec-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% ofc +c ₁ in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromesc +c ₁ (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromesc +c ₁. Addition of ferrocyanide to intact cytochromec-depleted mitochondria does not reduce cytochromec ₁; treatment withN,N,N,N-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromesc +c ₁. TheK _m value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochromec ₁ is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochromec ₁ is exposed to the matrix face and cytochromec is exposed to the cytoplasmic face. No redox center other than cytochromec in the segment between the antimycin site and cytochromec is exposed on the C-side.Abbreviations Used: MES, 2(N-morpholino)-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; TMPD,N,N,N,N-tetramethylphenylenediamine; ETP, electron transport particles; NAD-NADH, nicotinamide adenine dinucleotide; PMS, phenazine methosulfate.     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

Quantitative conformational analysis of cytochromec bound to phospholipid vesicles studied by resonance Raman spectroscopy

Resonance Raman spectra have been recorded from ferri-cytochromec bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG) or dioleoyl phosphatidylglycerol-dioleoyl phasphatidylcholine (DOPG-OPC) (70 : 30 mole/mole). Lipid binding induces very significant conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from two different conformational species, I and II, of the protein, and from two different spin and coordination states of the heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could be quantitated by a constraint interative fitting routine using single Lorentzian profiles. In the high frequency, or marker band region (1200 to 1700 cm⁻¹), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface enhanced resonance Raman measurements on cytochromec adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents of the different species. In the low frequency, or finger-print, region (200 to 800 cm⁻¹), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction between the spectra of ferri-cytochromec in the three different lipid systems, using the relative proportions of the species derived from the marker band region. These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented. The proportion of state II, in which hence the heme crevice assumes an open structure, and of the five-coordinated high spin configuration, is found to increase on binding ferri cytochromec to negatively charged lipid vesicles. The extent of this conformational change increases in the order: DOPG-DOPC<DOPG<DMPG, with a parallel decrease of the proportion of the conformational state I, whose structure is similar to that of the uncomplexed ferri-cytochrome c in solution. Similar conformational changes are found for ferro-cytochromec compared to those obtained with the oxidized species on binding to lipids. The present work is essential for studies which seek to analyze, in any detailed fashion, the conformational transitions in the heme protein which take place in response to changes in the lipid environment.