酸性条件下两种马来酸-烯烃交替共聚物诱导细胞色素c的结构转换

在pH=2·9时,细胞色素c保持类天然的结构;和马来酸-烯烃交替共聚物作用后,细胞色素c的α-螺旋结构基本保持不变,但是三级结构被破坏.另一方面,在pH=2·1时,细胞色素c去折叠形成伸展的无规卷曲构象;马来酸-烯烃交替共聚物可以诱导酸变性的细胞色素c从无规卷曲构象转变为α-螺旋结构.在酸性溶液中,由于马来酸-异丁烯交替共聚物和细胞色素c之间更强的相互作用,其对蛋白质结构的影响大于马来酸-1-十四烯交替共聚物.相对于小分子,聚合物可以在低浓度条件下提供有利于蛋白质结构转换的微环境.     

Structure and activity transition of lysozyme on interacting with and releasing from polyelectrolyte with different hydrophobicity

The complexes of lysozyme with poly(isobutylene‐alt‐maleic acid) (PIMA) and poly(1‐tetradecene‐alt‐maleic acid) (PTMA) at pH 7.4 were characterized using static and dynamic light scattering. The electrostatic interaction of PIMA with lysozyme results in a loose complex structure, while the electrostatic and hydrophobic interactions between PTMA and lysozyme produce a compact complex structure. Lysozyme in PIMA complex particles remains a part of activity and structure analyzed by circular dichroism and substrate hydrolysis. However, lysozyme loses its tertiary structure and activity completely in PTMA complex. NaCl is more effective to dissociate PIMA–lysozyme complex by screening the electrostatic interaction, whereas GdHCl is more effective to dissociate PTMA–lysozyme complex by forming GdHCl–PTMA precipitates through extra hydrophobic interactions. In the release process, the denatured lysozyme molecules are able to avoid hydrophobic aggregation and fully regenerate their native structure and activity. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4681–4690, 2008     

Interaction of apo cytochrome c with sulfonated polystyrene nanoparticles

Stable nanoparticle dispersion in aqueous solutions was obtained with partially sulfonated polystyrene. The hydrophobic association of the backbone chains and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The size distribution of the sulfonated polystyrene particles in relation to concentration, degree of sulfonation and chain length, and pH was characterized by dynamic laser light-scattering. The structure and morphology of the particles were characterized with fluorescence and atom force microscopy. Highly sulfonated polystyrene particles can form large complex particles with positively charged protein, apo cytochrome c. Dynamic laser light-scattering and atom force microscopy studies show that the size and distribution of the complex particles depend on the relative amount of apo cytochrome c and sulfonated polystyrene. When sulfonated polystyrene is in excess, apo cytochrome c interacts with sulfonated polystyrene particles forming stable complexes and excessive sulfonated polystyrene particles bind to the periphery of the complexes preventing them from further aggregation. When apo cytochrome c is in excess, apo cytochrome c links the complexes forming much larger particles. Fluorescence study demonstrates that the hydrophobicity/hydrophility of the complex particles is relative to the ratio of apo cytochrome c and sulfonated polystyrene, degree of sulfonation, and pH. Apo cytochrome c not only can neutralize the negative charges on the surface of sulfonated polystyrene particles, but may also insert into the cores disrupting the original structure of sulfonated polystyrene particles.     

Cytochrome c–dimyristoylphosphatidylglycerol interactions studied by asymmetrical flow field-flow fractionation

Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c–lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.Electronic Supplementary Material Supplementary material is available for this article at     

Thermal unfolding of proteins probed by laser spray mass spectrometry

The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.     

Weak interactions and molecular recognition in systems involving electron transfer proteins

Electrostatic interactions and other weak interactions between amino acid side chains on protein surfaces play important roles in molecular recognition, and the mechanism of their intermolecular interactions has gained much interest. We established that charged peptides are useful for investigating the molecular recognition character of proteins and their molecular interaction induced structural changes. Positively charged lysine peptides competitively inhibited electron transfer from reduced cytochrome f (cyt f or cytochrome c (cyt c) to oxidized plastocyanin (PC), due to neutralization of the negatively charged site of PC by formation of PC-lysine peptide complexes. Lysine peptides also inhibited electron transfer from cyt c to cytochrome c peroxidase. Likewise, negatively charged aspartic acid peptides interacted with the positively charged sites of cytfand cyt c, and competitively inhibited electron transfer from reduced cytfor cyt c to oxidized PC and from [Fe(CN)6]4- to oxidized cyt c. Changes in the geometry and a shift to a higher redox potential of the active site Cu of PC on oligolysine binding were detected by spectroscopic and electrochemical measurements, owing to the absence of absorption in the visible region for lysine peptides. Structural and redox potential changes were also observed for cyt f and cyt c by interaction with aspartic acid peptides.     

Probing a complex of cytochrome c and cardiolipin by magnetic circular dichroism spectroscopy: implications for the initial events in apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH(-)-ligated. The ferrous state is predominantly high-spin and, most likely, His/-. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form.     

Studies on the electrochemical behavior of cytochrome c and its interaction with DNA at a Co/GC ion implantation modified electrode

The electrochemical behavior of cytochrome c (cyt c) and its interaction with DNA at a Co/glassy carbon (GC) ion implantation modified electrode were studied by linear sweep and cyclic voltammetry. In 0.005 mol dm(-3) Tris-0.05 mol dm(-3) NaCl buffer solution (pH = 7.10), a sensitive reduction derivative peak of cyt c was obtained by linear sweep voltammetry. The peak potential was 0.032 V (SCE). The peak current was proportional to the concentration of cyt c. The electrode process was quasi-reversible with adsorption. The electrode reaction rate constant k and the electron transfer coefficient a of cyt c were 4.42 s(-1) and 0.47, respectively. AES and XPS experiments showed that Co was implanted into the surface of the GC electrode (GCE). The implanted Co formed Co-C, which catalyzed the reduction of cyt c. The reaction of DNA with cyt c led to an electrochemically active complex, which resulted in an increase in the reduction current of cyt c. After adding DNA into the solution containing cyt c, the electrode process was still quasi-reversible with adsorption.     

Reversible Electrochemistry of Cytochrome c on Recompressed,Binderless Exfoliated Graphite Electrodes

The direct electrochemistry of cytochrome c (cyt‐c) has been investigated on exfoliated graphite (EG) electrodes. The as‐polished and roughened (using SiC emery sheet) EG surfaces are inactive for the direct electron transfer. However, when the EG electrode was sonicated before the experiment, a pair of redox waves were obtained for freely diffusing cyt‐c in the solution phase. The formal potential was found to be 0.01 V (vs. SCE) in 0.1 M phosphate buffer at a pH of 7.1. The electrochemical response for the adsorbed cyt‐c on sonicated EG electrodes, which is shown to have carbonyl functional groups on its surface, shows nearly reversible voltammograms in the same electrolyte. However, the formal potential in the adsorbed state is more negative than that observed for the solution phase cyt‐c. A structure based on an open heme conformation proposed by Hildebrandt and Stockburger is probably present on the EG surface. It is suggested that the electrochemistry at the EG electrode is essentially governed by favourable electrostatic interactions.     

Effect of the electrostatic interaction on the redox reaction of positively charged cytochrome C adsorbed on the negatively charged surfaces of acid-terminated alkanethiol monolayers on a Au(111) electrode

The electrochemical properties of cytochrome c (cyt c) adsorbed on mixed self-assembled monolayers (SAMs) of 2-mercaptoethanesulfonate (MES)/2-mercaptoethanol (MEL) are compared with those on single-component SAMs of MES, MEL, and mercaptopropionic acid (MPA), using cyclic voltammetry and potential-modulated UV-vis reflectance spectroscopy. The rate constant of electron transfer (ET), k(et), of cyt c adsorbed on the SAM of MPA decreases from 1450 +/- 210 s(-1) at pH 7 to 890 +/- 100 s(-1) at pH 9. In contrast, the value of k(et) of cyt c on the SAM of MES is pH-independent at 100 +/- 15 s(-1). Those facts suggest that a large negative charge density on the SAM surface slows down the ET between cyt c and the electrode. The surface charge density of the SAM affects also the amount of electroactive cyt c, Gamma(e), which decreases from 10.0 +/- 1.0 to 5.3 +/- 1.1 pmol cm(-2) with increasing pH from 7 to 9 on the SAM of MPA. Similarly, the k(et) of cyt c adsorbed on the mixed SAMs of MES/MEL sharply decreases from 900 +/- 300 s(-1) to 110 s(-1) as the surface mole fraction of MES increases beyond 0.5, suggesting the presence of a negative surface charge threshold beyond which the rate of ET of cyt c is dramatically lowered. The decrease in the k(et) on the SAMs at high negative charge densities probably results from the confinement of adsorbed cyt c by the strong electrostatic force to an orientation that is not optimal for the ET reaction.     

Fe-Al-Mg-MMH/钠质蒙脱土分散体系触变性研究

研究了Fe-Al-Mg型混合金属氢氧化物(简称MMH)/钠质蒙脱土(简称MT)分散体系的触变性,发现在不同MMH/MT质量比(R)条件下,分散体系可分别呈现正触变性和负触变性.在所研究的R范围(0～0.091)内,随R增大,体系的触变性发生正触变性-复合触变性-负触变性的转化.考察了高速剪切分散后,测定粘度变化前的静置时间(ts)和测定粘度时的剪切速率(DL)对触变性的影响.探讨了各种触变性产生的原因,提出了“分散粒子-空间连续网络结构-密实聚集体”和“分散粒子-密实聚集体-密实聚集体簇”机理,较合理地解释了观察到的复合触变现象.     

Electrochemical Quartz Crystal Microbalance Studies on Cytochrome c/Polyelectrolyte Multilayer Assemblies on Gold Electrodes

Polyelectrolyte multilayer assemblies containing proteins are of interest for applications such as sensors, bioreactors, and bioelectronics. A multilayer electrode was built up by the layer‐by‐layer strategy consisting of alternating layers of cytochrome c and poly(aniline sulfonic acid). The electrode showed a linear increase of redox active protein with the number of deposited layers. The principle of electrode preparation was transferred from needle electrodes to planar surfaces in order to further the understanding of electron transfer through the layer assembly by means of electrochemical quartz crystal microbalance studies. The deposition process was followed on‐line by detection of the frequency shift of the crystals and was found to be rather fast (minutes). The total mass deposited was found to correlate well with the electrochemical response of the immobilized cyt.c. Furthermore, the influence of the polyelectrolyte was investigated by addition of PSS to the PASA solution. The strong interaction of the former polyelectrolyte seemed to hinder the electron transfer although a multilayer formation was proved. Dilution of the protein solution with redox inactive apo‐cyt.c led to a strong decrease of the voltammetric signal, well beyond the percentage of apo‐cyt.c inside the assembly. Thus, arguments for an electron transfer via protein–protein interaction were found.     

Radical para-benzoic acid derivatives: transmission of ferromagnetic interactions through hydrogen bonds at long distances

Investigation of the transmission of magnetic interactions through hydrogen bonds has been carried out for two different benzoic acid derivatives which bear either a tert-butyl nitroxide (NOA) or a poly(chloro)triphenylmethyl (PTMA) radical moiety. In the solid state, both radical acids formed dimer aggregates by the complementary association of two carboxylic groups though hydrogen bonding. This association ensured that atoms with most spin density are separated from one another by more than 15 A. Thus, no competing through-space magnetic exchange interactions are expected in these dimers and, hence, they provide good models to investigate whether noncovalent hydrogen bonds play a role in the long-range transmission of magnetic interactions. The nature of the magnetic exchange interaction and their strengths within similar dimer aggregates in solution was assessed by electron spin resonance (ESR) spectroscopy. In the case of radical NOA, low-temperature ESR experiments showed a weak ferromagnetic interaction between the two radicals in the dimer aggregates (which have the same geometry as in the solid state). In contrast, the corresponding solution ESR study performed with radical PTMA did not lead to any conclusive results, as aggregates were formed by noncovalent interactions other than hydrogen bonds. However, the bulkiness of the poly(chloro)triphenylmethyl radical prevented interdimer contacts in the solid state between regions of high spin density. Hence, solid-state measurements of the alpha phase of PTMA radical provided evidence of the intradimer interaction to confirm the transmission of a weak ferromagnetic interaction through the carboxylic acid bridges, as found for the NOA radical. Moreover, crystallization of the PTMA radical in presence of ethanol to form the beta phase of PTMA radical prevented the dimer formation; this resulted in the suppression of this interaction and provides further evidence of the magnetic exchange mechanism through noncovalent hydrogen bonds at long distances.     

Dynamic docking and electron transfer between Zn-myoglobin and cytochrome b(5)

We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4). The rates of photoinitiated ET between the Mb's (1-3) and cyt b(5) have been measured over a range of pH values and ionic strengths. Isothermal titration calorimetry (ITC) and NMR methods have been used to independently investigate the effect of charge neutralization on Mb/b(5) binding. The neutralization of the two heme propionates of ZnMb by formation of the heme diester or, for the first time, the diamide increases the second-order rate constant of the ET reaction between ZnMb and cyt b(5) by as much as several 100-fold, depending on pH and ionic strength, while causing negligible changes in binding affinity. Brownian dynamic (BD) simulations and ET pathway calculations provide insight into the protein docking and ET process. The results support a new "dynamic docking" paradigm for protein-protein reactions in which numerous weakly bound conformations of the docked complex contribute to the binding of cyt b(5) to Mb and Hb, but only a very small subset of these are ET active, and this subset does not include the conformations most favorable for binding; the Mb surface is a large "target" with a small "bullseye" for the cyt b(5) "arrow". This paradigm differs sharply from the more familiar, "simple" docking within a single, or narrow range of conformations, where binding strength and ET reactivity increase in parallel. Likewise, it is distinct from, although complementary to, the well-known picture of conformational control of ET through "gating", or a related picture of "conformational coupling". The new model describes situations in which tight binding does not correlate with efficient ET reactivity, and explains how it is possible to modulate reactivity without changing affinity. Such "decoupling" of reactivity from binding clearly is of physiological relevance for the reduction of met-Mb in muscle and of met-Hb in a red cell, where tight binding of cyt b(5) to the high concentration of ferrous-Mb/Hb would prevent the cytochrome from finding and reducing the oxidized proteins; it likely is of physiological relevance in other situations, as well.     

Mg-Fe-MMH-钠质蒙脱土分散体系的触变性研究

触变性是分散体系一种复杂的流变学性质,指流变性质随剪切时间而发生变化的现象。以往研究过程中,先后发现了切稀现象和切稠现象,即正触变性和负触变性。最近,我们又发现了复合触变性,即一个体系可先后呈现正触变性和负触变性特征。研究了Mg-Fe型混合金属氢氧化物(简称MMH)-钠质蒙脱土(简称MT)分散体系的触变性,发现在不同MMH/MT质量比条件下,可分别呈现正触变性、负触变性,另外双观察到了复合触变性。在所研究的MMH/MT质量比范围(0～0.5)内,随MMH/MT质量比增大,体系的触变性发生负触变性-正触变性-复合触变性-负触变性的转化。考察了电解质NaCl对触变性的影响,发现不改变触变性的类型。探讨了各种触变性产生的原因,提出了“分散粒子-空间连续网络结构-密实聚集体”机理,较合理地解释了观察到的复合触变现象。     

Real-time atomic force microscopy reveals cytochrome c-induced alterations in neutral lipid bilayers

The interaction of cytochrome c (cyt c) with supported lipid membranes was investigated on the nanoscale by real-time atomic force microscopy. Cyt c promoted the formation and the expansion of depressed areas in the fluid parts of the bilayer. When the depressions reached the gel domains, they induced the thickening of their edges. According to the step-height differences, cyt c was able to remove neutral lipids in the fluid phase and then to reside on the mica surface. Concerning gel phases, cyt c might insert between the two lipid leaflets, or it might intercalate between the mica and the bilayer.     

Oscillatory interaction between two like‐charged nanoparticles induced by polyelectrolyte brush–solvent interface

We use two‐dimensional (2D) self‐consistent field theory to study the effective interactions between two like‐charged cylindrical nanoparticles mediated by an oppositely weakly charged polyelectrolyte brush in a solvent solution. In a poor solvent, where a sharp brush–solvent interface forms, an oscillatory interaction is observed when two nanoparticles are both located at the brush–solvent interface. This oscillatory interaction depends on the penetration depths of the particles and their geometric orientations with respect to the substrate. When the particles are both immersed in the brush and/or the particles are oriented vertically or diagonally with large angles to the substrate, the oscillatory behavior disappears. We interpret our findings by analyzing in detail the contributions to the free energy from electrostatic interaction, nonelectrostatic interaction, and entropies, separately. Briefly, the deformations of the interface and the ion layers formed in the vicinity of the interface are responsible for this oscillatory behavior. In a good solvent, where the narrow brush–solvent interface vanishes, the effective particle–particle interactions behave like that for both particles immersed into the brush with poor solvent. They are found to be repulsive. The influences of the particle size, grafting density, and amount of charges and ions are also briefly discussed. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016 , 54, 1458–1468     

Factors determining the enzyme catalytic power caused by noncovalent interactions: Charge alterations in enzyme active sites

Understanding the origin of the enormous catalytic power of enzymes is very important. Electrostatic interactions and desolvation are the phenomena that are most proposed to explain the catalysis of enzymes; however, they also decelerate enzymatic reactions. How enzymes catalyze reactions through noncovalent interactions is still not well-understood. In this study, we explored how enzyme-substrate noncovalent interactions affect the free energy barriers (ΔG³s) of reactions by using a theoretical derivation approach. We found that enzymes reduce ΔG³s of reactions by decreasing positive charges and/or increasing negative charges in the electron-donating centers and by decreasing negative charges and/or increasing positive charges in the electron-accepting centers of reactions. Enzyme-substrate noncovalent interactions are essential approaches through which the charge alterations lead to ΔG³ reductions. Validations with reported experimental data demonstrated that this charge alteration mechanism can explain the catalyses caused by diverse types of noncovalent interactions. Electrostatic interactions and desolvation are the most observed noncovalent interactions essential for ΔG³ reductions. This mechanism does not contradict any specific enzymatic catalysis and overcomes the shortages of the electrostatic interaction and desolvation mechanisms. This study can provide useful guidance in exploring enzymatic catalysis and designing catalyst.     

Effect of mono-CDNP substitution of lysine residues on the redox reaction of cytochrome c electrostatically adsorbed on a mercaptoheptanoic acid modified Au(111) surface

The effect of charge-inverting modification of single surface lysine residue on the electron transfer (ET) reaction of horse heart cytochrome c (cyt c) is examined for 12 different types of mono-4-chloro-2,5-dinitrobenzoic acid substituted cyt c (mCDNPc) adsorbed on a Au(111) electrode modified with a self-assembled monolayer (SAM) of 7-mercapto-heptanoic acid (MHA). A negative shift in the redox potential by 10-35 mV as compared to that of native cyt c and a monolayer coverage in the range of 13-17 pmol cm(-2) are observed for electroactive mCDNPc's. The magnitude of the decrease in the ET rate constant (k(et)) of mCDNPc's compared with that of native cyt c depends on the position of the CDNP substitution. For mCDNPc's in which the modified lysine residue is outside of the interaction domain of cyt c with the SAM, the ratio of the k(et) of mCDNPc to that of native cyt c is correlated to the change in the dipole moment vector of cyt c due to the CDNP modification. This correlation suggests that the dipole moment of cyt c determines its orientation of adsorption on the SAM of MHA and significantly affects the rate of the ET. The CDNP modification of lysine residues at the interaction domain significantly decreases the rate, demonstrating the importance of the local charge environment in determining the rate of ET.     

Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.