Methionine ligand lability of type I cytochromes c: detection of ligand loss using protein film voltammetry

Protein film voltammetry (PFV) is used to interrogate the behavior of a variety of bacterial and mitochondrial His/Met-ligated cytochromes c. While analogous studies upon alkanethiol-modified gold electrodes reveal the anticipated Fe(II/III) couple only, PFV using pyrolytic graphite edge (PGE) electrodes demonstrates the presence of a lower-potential form of each of the cyts c studied, with a potential of approximately -100 mV (vs hydrogen). The generation of the novel, lower-potential state is shown to arise specifically from the interaction with the PGE electrode. Simultaneously, the typical Fe(II/III) couple can be observed. PFV of a series of wild-type cytochromes and mutants in the Met-donating loop show that the lower-potential state is highly similar between proteins from Pseudomonas aeruginosa (PA), Hydrogenobacter thermophilus (HT), and horse heart. The generation of the lower-potential form correlates inversely with the stability of the Met-Fe interaction for each of the cytochromes. Comparison with chemically unfolded cyts c indicates that the lower-potential forms detected here are unique, and this distinct state is ascribed to the loss of the Met ligand. Thus, PGE is demonstrated to be a non-innocent electrode surface in PFV studies of His/Met-ligated cytochromes c.     

Heme axial methionine fluxion in Pseudomonas aeruginosa Asn64Gln cytochrome c551

Heme axial methionine ligands in ferricytochromes c552 from Hydrogenobacter thermophilus (HT) and Nitrosomonas europaea, both members of the cyt c8 family, display fluxional behavior. The ligand motion, proposed to be inversion at sulfur, results in an unusually small range of hyperfine shifts for heme substituents in these proteins. Herein, heme axial Met fluxion is induced in a structurally homologous cytochrome c551 from Pseudomonas aeruginosa (PA) by substituting heme pocket residue Asn64 with Gln. The mutant, PA-N64Q, displays a highly compressed range of heme substituent hyperfine shifts, temperature-dependent heme methyl resonance line broadening, low rhombic magnetic anisotropy, and a magnetic axes orientation consistent with Met orientational averaging. Analysis of NMR properties of PA-N64Q demonstrates that the heme pocket of the mutant resembles that of HT. This result confirms the importance of peripheral interactions and, in particular, residue 64 in determining axial Met orientation and heme electronic structure in proteins in the cyt c8 family.     

Modulation of the ligand-field anisotropy in a series of ferric low-spin cytochrome c mutants derived from Pseudomonas aeruginosa cytochrome c-551 and Nitrosomonas europaea cytochrome c-552: a nuclear magnetic resonance and electron paramagnetic resonance study

Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.     

Spectroscopic characterization and assignment of reduction potentials in the tetraheme cytochrome C554 from Nitrosomonas europaea

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is an essential electron transfer component in the biological oxidation of ammonia. The protein contains one 5-coordinate heme and three bis-His coordinated hemes in a 3D arrangement common to a newly characterized class of multiheme proteins. The ligand binding, electrochemical properties, and heme-heme interactions are investigated with M?ssbauer and X- and Q-band (parallel/perpendicular mode) EPR spectroscopy. The results indicate that the 5-coordinate heme will not bind the common heme ligands, CN(-), F(-), CO, and NO in a wide pH range. Thus, cyt c(554) functions only in electron transfer. Analysis of a series of electrochemically poised and chemically reduced samples allows assignment of reduction potentials for heme 1 through 4 of +47, +47, -147, and -276 mV, respectively. The spectroscopic results indicate that the hemes are weakly exchange-coupled (J approximately -0.5 cm(-)(1)) in two separate pairs and in accordance with the structure: hemes 2/4 (high-spin/low-spin), hemes 1/3 (low-spin/low-spin). There is no evidence of exchange coupling between the pairs. A comparison of the reduction potentials between homologous hemes of cyt c(554) and other members of this new class of multiheme proteins is discussed. Heme 1 has a unique axial N(delta)-His coordination which contributes to a higher potential relative to the homologous hemes of hydroxylamine oxidoreductase (HAO) and the split-Soret cytochrome. Heme 2 is 300 mV more positive than heme 4 of HAO, which is attributed to hydroxide coordination to heme 4 of HAO.     

Cytochrome c552 mutants: structure and dynamics at the active site probed by multidimensional NMR and vibration echo spectroscopy

Spectrally resolved infrared stimulated vibrational echo experiments are used to measure the vibrational dephasing of a CO ligand bound to the heme cofactor in two mutated forms of the cytochrome c552 from Hydrogenobacter thermophilus. The first mutant (Ht-M61A) is characterized by a single mutation of Met61 to an Ala (Ht-M61A), while the second variant is doubly modified to have Gln64 replaced by an Asn in addition to the M61A mutation (Ht-M61A/Q64N). Multidimensional NMR experiments determined that the geometry of residue 64 in the two mutants is consistent with a non-hydrogen-bonding and hydrogen-bonding interaction with the CO ligand for Ht-M61A and Ht-M61A/Q64N, respectively. The vibrational echo experiments reveal that the shortest time scale vibrational dephasing of the CO is faster in the Ht-M61A/Q64N mutant than that in Ht-M61A. Longer time scale dynamics, measured as spectral diffusion, are unchanged by the Q64N modification. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to confirm that the dynamical difference induced by the Q64N mutation is primarily an increase in the fast (hundreds of femtoseconds) frequency fluctuations, while the slower (tens of picoseconds) dynamics are nearly unaffected. We conclude that the faster dynamics in Ht-M61A/Q64N are due to the location of Asn64, which is a hydrogen bond donor, above the heme-bound CO. A similar difference in CO ligand dynamics has been observed in the comparison of the CO derivative of myoglobin (MbCO) and its H64V variant, which is caused by the difference in axial residue interactions with the CO ligand. The results suggest a general trend for rapid ligand vibrational dynamics in the presence of a hydrogen bond donor.     

Control of cytochrome C redox potential: axial ligation and protein environment effects

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.     

Modulation of heme redox potential in the cytochrome c6 family

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6 but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c6 despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c6 from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c6A. One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c6 is replaced by Val (V52) in c6A. Using protein film voltammetry, we found that swapping these residues raised the c6A potential by +109 mV and decreased that of c6 by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c6 and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c6A from cytochrome c6, inhibiting reduction by the cytochrome b6f complex and facilitating establishment of a new function.     

Roles of the proximal hydrogen bonding network in cytochrome P450cam-catalyzed oxygenation

Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I. J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause approximately 45 and approximately 35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482 approximately 483 cm(-)(1) compared with that of wild-type P450(cam) at 484 cm(-)(1). The Q360 mutants also showed the upshift by 4 approximately 5 cm(-)(1) of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the Q360 mutants and were in contrast to the increased pi-back-donation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-)(1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.     

Electron transfer and electrocatalytic properties of the immobilized methionine80alanine cytochrome c variant

The M80A variant of yeast iso-1-cytochrome c (cytc), which features a noncoordinating Ala residue in place of the axial heme iron Met ligand, was chemisorbed on a gold electrode coated with 4-mercaptopyridine or carboxyalkanethiol self-assembled monolayers (SAM) and investigated by cyclic voltammetry at varying conditions of temperature, pH, and O2 concentration. The E degrees ' value (standard reduction potential for the heme Fe(III)/Fe(II) couple) of M80A cytc on both SAMs is of approximately -200 mV (vs the standard hydrogen electrode, SHE) at pH 7, which is more than 400 mV lower than that of native cytochrome c in the same conditions. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of heterogeneous electron transfer (ET) are dominated by the presence of a hydroxide ion as the sixth axial heme iron ligand above pH 6. On both SAMs, protonation of the bound hydroxide ion is mainly responsible for the changes in these parameters at low pH, since the distances of ET between the heme and the electrode are found to be independent of pH in the range of 5-11. The invariance of the electrochemical features up to pH 11 indicates that no changes in heme iron coordination occur at high pH, at variance with native cytc. Most notably, immobilized M80A cytc is found to act as an efficient biocatalyst for O2 reduction from pH 5 to 11.0. This finding makes M80A cytc a suitable candidate as a constituent of a biocatalytic interface for O2 biosensing and opens the way for the exploitation of engineered cytochrome c in the bio-based detection of chemicals of environmental and clinical interest.     

The redox chemistry of the covalently immobilized native and low-pH forms of yeast iso-1-cytochrome c

Cyclic voltammetry experiments were carried out on native Saccharomyces cerevisiae iso-1-cytochrome c and its C102T/N62C variant immobilized on bare polycrystalline gold electrode through the S-Au bond formed by a surface cysteine. Experiments were carried out at different temperatures (5-65 degrees C) and pH values (1.5-7). The E degrees ' value at pH 7 (+370 mV vs SHE) is approximately 100 mV higher than that for the protein in solution. This difference is enthalpic in origin and is proposed to be the result of the electrostatic repulsion among the densely packed molecules onto the electrode surface. Two additional electrochemical waves are observed upon lowering the pH below 5 (E degrees ' = +182 mV) and 3 (E degrees ' = +71 mV), which are attributed to two conformers (referred to as "intermediate" and "acidic", respectively) featuring an altered heme axial ligation. This is the first determination of the reduction potential for low-pH conformers of cytochrome c in the absence of denaturants. Since the native form of cytochrome c can be restored, bringing back the pH to neutrality, the possibility offered by this transition to reversibly modulate the redox potential of cytochrome c is appealing for bioelectronic applications. The immobilized C102T/N62C variant, which differs from the native protein in the orientation of the heme group with respect to the electrode, shows very similar reduction thermodynamics. For both species, the rate constant for electron transfer between the heme and the electrode increases for the acidic conformer, which is also found to act as a biocatalytic interface for dioxygen reduction.     

Engineering artificial redox chains by molecular 'Lego'

This work reports on a novel approach for building artificial redox chains: the molecular 'Lego' approach. This exploits the scaffold of natural redox proteins by fusing together functional protein modules with the desired properties. The molecular 'Lego' mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments. Non-physiological electron transfer partners, flavodoxin (fld) and cytochrome c553 (c553) from Desulfovibrio vulgaris and the haem domain of P450 BM3 (BMP) from Bacillus megaterium have been used as building blocks in different combinations to build artificial redox chains. The kinetic characterization of the electron transfer (ET) between the separate building blocks has been carried out. Under pseudo-first order conditions, a limiting ET rate, klim, of 0.48 +/- 0.05 s-1 and 43.77 +/- 2.18 s-1 and an apparent binding constant, Kapp, of 21 +/- 6 microM and 1.23 +/- 0.32 microM have been found for the fld/c553 and fld/BMP redox pairs, respectively. These results show that fld can be used as a module for transferring electrons to c553 and BMP. A 3D model of the fld/c553 and fld/BMP complexes was used to guide the construction of covalently linked assemblies via engineered disulfide bridges or by fusion of the relevant genes via an engineered loop. The first approach led to the construction, expression and characterization of the S35C and S64C mutants of fld and M23C and G51C mutants of c553. Although the redox potentials of the separate mutants were found to be the same as those of recombinant wild type proteins (-408 mV for the semiquinone/hydroquinone couple of fld and +32 mV for the c553), the c553 homo-dimers M23C-M23C and G51C-G51C were found to have redox potentials of +88 and +105 mV, respectively. These differences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox potentials of the transient redox complexes in physiological systems. The fld-c553 S64C-M23C and S35C-M23C chimeras were constructed, expressed and purified but the FMN was found to be destabilised resulting in the apo-form of these proteins. The gene fusion strategy was used to produce covalently linked assemblies of both fld-c553 and fld-BMP. The former was expressed using a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c553. The fld-BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471-479) to link the BMP domain at the N-terminus with fld domain at the C-terminus. This fusion was found to be correctly folded and functional. Efficient ET from the FMN to the haem domain (370 s-1) was also found to be in the same region of the physiological redox partners (250 s-1). This work demonstrates the feasibility of the molecular 'Lego' approach in generating functional multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.     

Effects of heme on the thermal stability of mesophilic and thermophilic cytochromes c: comparison between experimental and theoretical results

We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25?°C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c(551), PH c(552), HT c(552), and AA c(555)) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c, the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c(555). However, AA c(555) has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c(555) is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c(555) has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25?°C the α-helix content of the apo state of AA c(555) is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c(551), PH c(552), and HT c(552)).     

Control of the stability of Hydrogenobacter thermophilus cytochrome C(552) through alteration of the basicity of the N-terminal amino group of the polypeptide chain

In the denatured state of Hydrogenobacter thermophilus cytochrome c(552) (HT), the N-terminal amino group of the polypeptide chain is coordinated to the heme Fe in place of the axial Met, the His-N(term) form being formed [Tai, H., Munegumi, T., Yamamoto, Y. Inorg. Chem. 2009, 48, 331-338]. Since the His-N(term) form can be considered as an ordered residual structure in the denatured protein, its stability significantly influences the energy of the denatured state. In this study, the His-N(term) forms of the wild-type HT and its mutants possessing a series of amino acid residues at the N-terminal, such as N1D, N1E, and N1G, have been characterized to elucidate the physicochemical properties of the N-terminal residue responsible for the control of the thermodynamic stability of the His-N(term) form. The study revealed that the thermodynamic stability of the His-N(term) form depends highly on the basicity of the N-terminal amino group of the polypeptide chain in such a manner that an increase in the pK(a) value of the N-terminal amino group by 1 unit results in stabilization of the bond between heme Fe and the N-terminal amino group (Fe-N(term) bond) in the His-N(term) form by ～4 kJ mol(-1). The empirical hard and soft acid and base principle could account for the observed relationship between the pK(a) value of the N-terminal amino group and the stability of the Fe-N(term) bond in the His-N(term) form. In addition, the study demonstrated that the overall stability of the protein can be manipulated through the energy of the denatured protein by changing the thermodynamic stability of the His-N(term) form. Consequently, the overall stability of the protein has been shown to be controlled through alteration of the basicity of the N-terminal amino group of the polypeptide chain. These findings provide new insights into the stabilizing interactions in the denatured protein, which are relevant as to characterization of the protein stability and folding.     

Contributions to cytochrome c inner- and outer-sphere reorganization energy

Cytochromes c are small water-soluble proteins that catalyze electron transfer in metabolism and energy conversion processes. Hydrogenobacter thermophilus cytochrome c₅₅₂ presents a curious case in displaying fluxionality of its heme axial methionine ligand; this behavior is altered by single point mutation of the Q64 residue to N64 or V64, which fixes the ligand in a single configuration. The reorganization energy (λ) of these cytochrome c₅₅₂ variants is experimentally determined using a combination of rotating disc electrochemistry, chronoamperometry and cyclic voltammetry. The differences between the λ determined from these complementary techniques helps to deconvolute the contribution of the active site and its immediate environment to the overall λ (λ_Total). The experimentally determined λ values in conjunction with DFT calculations indicate that the differences in λ among the protein variants are mainly due to the differences in contributions from the protein environment and not just inner-sphere λ. DFT calculations indicate that the position of residue 64, responsible for the orientation of the axial methionine, determines the geometric relaxation of the redox active molecular orbital (RAMO). The orientation of the RAMO with respect to the heme is key to determining electron transfer coupling (H_AB) which results in higher ET rates in the wild-type protein relative to the Q64V mutant despite a 150 mV higher λ_Total in the former.

Efficient delocalization of the redox-active molecular orbital (RAMO) in HtWT results in an increase in H_AB value which in turn accelerates the electron transfer (ET) rate in spite of the higher reorganization energy (λ) than the HtQ64V mutant.     

Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes

Shewanella oneidensis MR‐1 gains energy by extracellular electron transfer to solid surfaces. They employ c‐type cytochromes in two Mtr transmembrane complexes, forming a multiheme wire for electron transport across the cellular outer membrane. We investigated electron‐ and hole‐transfer mechanisms in the external terminal of the two complexes, MtrC and MtrF. Comparison of computed redox potentials with previous voltammetry experiments in distinct environments (isolated and electrode‐bound conditions of PFV or in vivo) suggests that these systems function in different regimes depending on the environment. Analysis of redox potential shifts in different regimes indicates strong coupling between the hemes via an interplay between direct Coulomb and indirect interactions through local structural reorganization. The latter results in the screening of Coulomb interactions and explains poor correlation of the strength of the heme‐to‐heme interactions with the distance between the hemes.     

Evaluation of osmium(II) complexes as electron transfer mediators accessible for amperometric glucose sensors.

In order to lower the redox potentials of Os(III/II) complexes, the mixed ligand complexes of Os(II) were synthesized. The redox potentials of Os(III/II) complexes could be lowered by the use of 4,4'-dimethyl-2,2'-bipyridine (dmbpy), imidazole (Him) or its derivatives, and chloride ion as ligands, e.g., values of the redox (formal) potentials of 628 mV vs. Ag/AgCl for [Os(bpy)3]3+/2+ (bpy: 2,2'-bipyridine) and -6 mV for [OsCl(Him)(dmbpy)2]2+/+ were given in deaerated 0.1 mol dm-3 phosphate buffer (pH 7.0). The evaluation of Os(II) complexes as electron transfer mediators accessible for amperometric glucose sensors was examined according to the determination of the redox potentials of Os(III/II) complexes and the second-order rate constants for electron transfer between glucose oxidase (GOx) in reduced form and the Os(III) complex. Although the Os(II) complexes with lower redox potentials tended to decrease the second-order rate constants ks, the ks values for the majority of Os(II) complexes synthesized in this study were greater than that for ferrocenecarboxylic acid. Acceleration of the electron-transfer reaction is attributable to the hydrogen bonding and/or the electrostatic interaction between the Os(II) complexes and GOx. It may be consequently concluded that the mixed ligand complexes of Os(II) with bpy (dmbpy), Him (its derivatives), and Cl- can act as more efficient electron transfer mediators for the fabrication of amperometric glucose sensors.     

径向电场调制毛细管电泳法用于神经递质分离

利用双向电场控制毛细管电泳系统，考察了神经递质的分离。在pH2.5的0.01mol/L磷酸盐缓冲体系中，通过加入20％（V／V）正丙醇，改善了多巴胺和5－羟色胺的分离效果，但仍不太理想。通过施加径向电场，可进一步提高分离度。本研究不仅拓宽了径向电场调控的样品分离范围，而且为生物活性物质的痕量分析提供了参考依据。     

Myoglobin-CO substate structures and dynamics: multidimensional vibrational echoes and molecular dynamics simulations

Spectrally resolved infrared stimulated vibrational echo data were obtained for sperm whale carbonmonoxymyoglobin (MbCO) at 300 K. The measured dephasing dynamics of the CO ligand are in agreement with dephasing dynamics calculated with molecular dynamics (MD) simulations for MbCO with the residue histidine-64 (His64) having its imidazole epsilon nitrogen protonated (N(epsilon)-H). The two conformational substate structures B(epsilon) and R(epsilon) observed in the MD simulations are assigned to the spectroscopic A(1) and A(3) conformational substates of MbCO, respectively, based on the agreement between the experimentally measured and calculated dephasing dynamics for these substates. In the A(1) substate, the N(epsilon)-H proton and N(delta) of His64 are approximately equidistant from the CO ligand, while in the A(3) substate, the N(epsilon)-H of His64 is oriented toward the CO, and the N(delta) is on the surface of the protein. The MD simulations show that dynamics of His64 represent the major source of vibrational dephasing of the CO ligand in the A(3) state on both femtosecond and picosecond time scales. Dephasing in the A(1) state is controlled by His64 on femtosecond time scales, and by the rest of the protein and the water solvent on longer time scales.     

Loop-contraction mutagenesis of type 1 copper sites

The shortest known type 1 copper binding loop (that of amicyanin, Ami) has been introduced into three different cupredoxin beta-barrel scaffolds. All of the loop-contraction variants possess copper centers with authentic type 1 properties and are redox active. The Cu(II) and Co(II) sites experience only small structural alterations upon loop contraction with the largest changes in the azurin variant (AzAmi), which can be ascribed to the removal of a hydrogen bond to the coordinating thiolate sulfur of the Cys ligand. In all cases, loop contraction leads to an increase in the pK(a) of the His ligand found on the loop in the reduced proteins, and in the pseudoazurin (Paz) and plastocyanin (Pc) variants the values are almost identical to that of Ami ( approximately 6.7). Thus, in Paz, Pc, and Ami, the length of this loop tunes the pK(a) of the His ligand. In the AzAmi variant, the pK(a) is 5.5, which is considerably higher than the estimated value for Az (<2), and other controlling factors, along with loop length, are involved. The reduction potentials of the loop-contraction variants are all lower than those of the wild-type proteins by approximately 30-60 mV, and thus this property of a type 1 copper site is fine-tuned by the C-terminal loop. The electron self-exchange rate constant of Paz is significantly diminished by the introduction of a shorter loop. However, in PcAmi only a 2-fold decrease is observed and in AzAmi there is no effect, and thus in these two cupredoxins loop contraction does not significantly influence electron-transfer reactivity. Loop contraction provides an active site environment in all of the cupredoxins which is preferable for Cu(II), whereas previous loop elongation experiments always favored the cuprous site. Thus, the ligand-containing loop plays an important role in tuning the entatic nature of a type 1 copper center.