Dynamics of the excited state of the primary electron donor in reaction centers of Rhodopseudomonas viridis as revealed by hole burning at 1.7K. Different conformational states

The spectra of absorbance changes (delta A) due to the formation of P+Q- (P, primary electron donor, Q, primary quinone acceptor) at 1.7K in Rhodopseudomonas viridis reaction centers (RCs) excited at 1014 nm has been shown to include, besides a progression of broad (170-190 cm-1) Gaussian vibronic bands separated by 150 cm-1, a 'narrow' structure near 1014 nm which can be simulated by a Lorentian zero-phonon hole (ZPH) and Lorentian one-mode (26.8 cm-1) phonon wings. The widths of ZPH of approximately 17 cm-1 for delta A reflecting the formation of P+Q- decaying in the ms time domain and of 6.8 +/- 0.4 cm-1 for P+Q- decaying in the min time domain at 1.7K, seems to correspond to different conformations of RCs with a relaxation time of P* of approximately 0.6 ps (in agreement with measurements in this time domain) and 1.6 +/- 0.1 ps, respectively. The comparison of the spectra of delta A in the region of the BL band for slow (min) and fast (ms) decaying components suggests a different mutual arrangement of P and BL for different conformations of RCs. It is assumed that the broad and narrow structures of the P band reflect the transitions to two configurations with different P-protein interactions. 'Narrow' structure of delta A spectrum with essentially the same phonon wings and ZPH (width of 3.8 +/- 0.4 cm-1) was observed within the P band when HL was photoreduced at 1.7K.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Resolution of low-energy chlorophylls in Photosystem I of Synechocystis sp. PCC 6803 at 77 and 295 K through fluorescence excitation anisotropy

Fluorescence excitation spectra of highly anisotropic emission from Photosystem I (PS I) were measured at 295 and 77 K on a PS II-less mutant of the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803). When PS I was excited with light at wavelengths greater than 715 nm, fluorescence observed at 745 nm was highly polarized with anisotropies of 0.32 and 0.20 at 77 and 295 K, respectively. Upon excitation at shorter wavelengths, the 745-nm fluorescence had low anisotropy. The highly anisotropic emission observed at both 77 and 295 K is interpreted as evidence for low-energy chlorophylls (Chls) in cyanobacteria at room temperature. This indicates that low-energy Chls, defined as Chls with first excited singlet-state energy levels below or near that of the reaction center, P700, are not artifacts of low-temperature measurements.If the low-energy Chls are a distinct subset of Chls and a simple two-pool model describes the excitation transfer network adequately, one can take advantage of the low-energy Chls' high anisotropy to approximate their fluorescence excitation spectra. Maxima at 703 and 708 nm were calculated from 295 and 77 K data, respectively. Upper limits for the number of low-energy Chls per P700 in PS I from S. 6803 were calculated to be 8 (295 K) and 11 (77 K).Abbreviations Chl - chlorophyll - BChl - bacteriochlorophyll - LHC - light-harvesting chlorophyll - PS - Photosystem - RC - reaction center - S. 6803 - Synechocystis sp. PCC 6803     

Triplet state magnetic resonance and fluorescence spectroscopy of metal-substituted hemoglobins.

Fluorescence detected magnetic resonance (FDMR) spectra detected at 596 nm of zinc-substituted hemoglobins at 4.2 K show a split D-E transition, which is not observed for zinc protoporphyrins ligated by methylimidazole in glasses. Incorporation of the zinc heme into the globin pocket is also accompanied by a blue shift of the fluorescence of 20 nm at 4.2 K. FDMR spectra recorded at 576 nm do not show the D-E splitting. The D-E splitting and the huge blue shift are not observed for the magnesium-substituted hemoglobins. Fluorescence measurements at 4.2 K and 77 K, and EPR measurements at 110 K, were carried out to obtain information about the ligation states of the zinc and magnesium protoporphyrins in glasses and in hemoglobin. The results are explained by considering ligation effects and distortion of the porphyrin plane.     

Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats

A new metabolite of vitamin K, 2(3)-hydroxy-2,3-dihydro-2-methyl,3-phytyl-1,4-naphthoquinone (hydroxyvitamin K), has been identified as a product of vitamin K epoxide metabolism in hepatic microsomes from warfarin-resistant rats, but not in those derived from normal rats. The structure was determined by comparison of the high performance liquid chromatography retention times, UV, IR, CD, and mass spectra of the unknown with chemically synthesized standards. Alterations in the formation of hydroxyvitamin K occur in parallel with alterations in total vitamin K epoxide conversion with respect to reaction time, extent of reaction, detergent stimulation, and inhibition by warfarin. Thus, hydroxyvitamin K appears to be a product of the warfarin-resistant vitamin K epoxide reductase. It is neither a substrate nor an inhibitor of epoxide reduction. Hydroxyvitamin K is formed from both enantiomers of racemic vitamin K epoxide with little stereoselectivity for the configuration of either the oxirane ring or the phytyl side chain. The reaction is stereospecific; however, the biologically formed (+)-vitamin K epoxide yields exclusively (+)-3-hydroxyvitamin K. Observation of this product is discussed as a key to understanding the normal reaction mechanism of the enzyme.     

Kinetic and spectroscopic evidence of cation-induced conformation changes in yeast K+ -activated aldehyde dehydrogenase.

The activity, stability and spectroscopic properties of yeast K+ -activated aldehyde dehydrogenase were measured at various times after removal from, and after returning to a solution containing K+. Enzyme activity is rapidly lost on removal of most of the K+ and rapidly regained if K+ is replaced immediately. These activity changes are slower than likely rates of K+ dissociation and association. These rapid changes in concentration result in altered enzyme stability with enzyme in K+ the more stable. U.v. difference spectra are produced whenever enzyme in an activating environment (K+ or Tl+) is compared with enzyme in a non-activating environment (Tris+ or Li+). These spectral changes occur within 10s. The saturation characteristics with K+ are hyperbolic for all three phenomena of activation, stabilization and spectral change, with estimated apparent dissociation constants (Ks) for K+ of 7.5 mM, 5.5 mM and 6 mM respectively. Continued incubation of enzyme in the absence of K+ results in the accumulation of an enzyme form that re-activates only slowly on replacing K+. Stability characteristics in various concentrations of K+ over equivalent time scales are consistent with the existence of additional conformations. Spectroscopic evidence also indicates such additional slow conformation changes. Results have been interpreted in terms of two separate conformation transitions induced or stabilized by K+.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

Measurement of extracellular pH,K(+), and lactate in ischemic heart

Simultaneous and continuous measurements of extracellular pH, potassium (K(+)), and lactate (L(-)) in ischemic rabbit papillary muscle are presented for the first time. Potentiometric pH and K(+) sensors and an amperometric lactate biosensor were used. These miniature electrodes were previously developed and individually tested for this purpose. The pH sensor was based on an iridium oxide layer electrodeposited on a planar platinum electrode fabricated on a flexible substrate. The potentiometric K(+) sensor was based on a polymeric membrane and valinomycin ionophore. The L(-) biosensor was based on lactate oxidase and an organic conducting salt polarized at 0.15V vs Ag/AgCl reference electrode. The utility of this novel analytical system to cardiovascular research was demonstrated by using the system to study the interrelationship of cellular K(+) and lactate loss in ischemic myocardium, and the role of extracellular pH and buffer capacity on this relationship. The results indicated: (i) sequential brief episodes of ischemia produced reproducible trends of L(-), pH, and K(+) changes during the first three episodes, (ii) extracellular L(-) increased with increasing buffer capacity of extracellular compartment, (iii) the patterns of extracellular L(-) and K(+) changes were not related directly, and (iv) L(-) transport and lactic acid diffusion were not the primary cause of extracellular acidosis during ischemia.     

A magnetic study of acidic ferric hemoglobin

In this article, we have extensively studied and discussed the magnetic properties of acidic ferric hemoglobin and its isolated chains. The magnetic susceptibility, EPR and optical spectra of those samples were measured in the temperature region below 77 degrees K. By the magnetic susceptibility measurements, it could be made clear that at an acidic pH value, both ferric hemoglobin and its isolated chains were constituted of a mixture of two spin states (high-spin state S = 5/2 and low-spin state S = 1/2) and the ratio of this mixture varied in each protein sample, but was independent of the temperature change below 77 degrees K. The co-existence of these two components could be ascertained by the observation of EPR spectra at liquid hydrogen temperature. Acidic ferric hemoglobin and its isolated chains exhibited the two components of EPR spectra which corresponded to their magnetic susceptibility, and it was found that the relaxation time of the low-spin state was longer than that of the high-spin state. The low-spin component of EPR spectra was almost undetectable at liquid nitrogen temperature. The three principal g values of this low-spin were gz = 2.80, gy = 2.20, and gx = 1.70. At alkaline pH values these low-spin components and the high-spin component of EPR spectra were displaced by the different low-spin spectra which corresponded to the ferric hemoglobin-hydroxide complex. It seems that the magnetic properties of the high-spin component are the same as the acidic ferric myoglobin, and the fine structure of the iron ion also seems to be same. Optical spectroscopy also gave similar magnetic properties which corresponded to the magnetic measurements.     

Primary photosynthetic processes in Heliobacterium chlorum at 15K

Absorbance changes induced by 25-ps laser flashes were measured in membranes of Heliobacterium chlorum at 15 K. Absorbance difference spectra, measured at various times after the flash showed negative bands in the Q_y region at 812, 793 and 665 nm. The first of these bands was attributed to the formation of excited singlet states of a long-wavelength form of antenna bacteriochlorophyll g (BChl g 808). Absorbance changes of shorter wavelength absorbing antenna BChls g were at least an order of magnitude smaller, indicating rapid excitation energy transfer (i.e. within the time resolution of the apparatus) from these BChls to BChl g 808. Excited BChl g 808 showed a bi-exponential decay with time constants of 50 and 200 ps. The bands at 793 and 665 nm may be attributed to the primary charge separation and reflect the photooxidation of the primary electron donor P-798 and photoreduction of a primary electron acceptor absorbing near 670 nm, presumably a BChl c or Chl a-like pigment. The bleaching of this pigment reversed with a time constant of 300 ps at 15 K and of 800 ps at 300 K. This indicates that electron transfer from the primary to the secondary electron acceptor is approximately 2.5 times faster at 15 K than at room temperature.Abbreviations BChl bacteriochlorophyll - FWHM full width at half maximum - P-798 primary electron donor - Tris tris(hydroxymethyl)amino methane     

Fluorescence and phosphorescence of adrenolutin

Adrenolutin 3,5,6-trihydroxy-1-methylindole is an intermediate in the metabolism of adrenaline and in the formation of adrenochromo-melanins. Excitation and emission spectra, quantum yield of the adrenolutin fluorescence in water, D2O, ethanol, methanol, acetone and aqueous phosphate buffer at different pH at 293K temperature are reported. Dependence of the quantum yield of adrenolutin on its concentration are measured. Lifetimes of 0.1 mM adrenolutin in water and ethanol are 32.0 +/- 0.2 ns and 9.2 +/- 0.2 ns respectively. Also fluorescence and phosphorescence spectra of adrenolutin in methanol at 110K are obtained. Degrees of polarization and angles between the dipoles for the three main bands absorption of adrenolutin from measurements at 103K are calculated. Adrenolutin may be classified as one of the most strongly fluorescing metabolites. Broad excitation spectrum and high quantum yields make this compound a potential effective acceptor of excitation energy.     

K+-selective channel from sarcoplasmic reticulum of split lobster muscle fibers

The patch clamp technique has been used to study channels in a membrane inside a cell. A single muscle fiber is skinned in relaxing saline (high K+, low Ca2+ with EGTA and ATP), leaving the native sarcoplasmic reticulum (SR) membrane exposed for patching. Fibers are dissected from the second antenna remotor muscles of the American lobster, Homarus americanus. Transmission and scanning electron microscopy confirm the large volume fraction of SR (approximately 70%) and absence of sarcolemma in this unusual skinned preparation. The resting potential of the SR was measured after the resistance of the patch of membrane was broken down. It is near 0 mV (-0.4 +/- 0.6 mV). The average input resistance of the SR is 842 +/- 295 M omega. Some 25% of patches contain a K+-selective channel with a mean open time of seconds and the channel displays at least two conducting states. The open probability is weakly voltage dependent, large at zero and positive potentials (cytoplasm minus SR lumen), and decreasing at negative potentials. The maximal conductance of this channel is 200 +/- 1 pS and the substate conductance is 170 +/- 3 pS in symmetrical 480 mM K+ solution. The current-voltage relation of the open channel is linear over a range of +/- 100 mV. The selectivity is similar to the SR K+ channel of vertebrates: PK/PNa is 3.77 +/- 0.03, determined from reversal potential measurements, whereas gamma K/gamma Na is 3.28 +/- 0.06, determined from open-channel conductance measurements in symmetrical 480 mM solutions. Voltage-dependent block in the lobster SR K+ channel is similar to, but distinct from, that reported for the vertebrate channels. It occurs asymmetrically when hexamethonium is added to both sides of the membrane. The block is more effective from the cytoplasmic side of the channel.     

Chlorophyll fluorescence emission spectroscopy of oxygenic organisms at 77 K

Photosynthetic fluorescence emission spectra measurement at the temperature of 77 K (–196°C) is an often-used technique in photosynthesis research. At low temperature, biochemical and physiological processes that modulate fluorescence are mostly abolished, and the fluorescence emission of both PSI and PSII become easily distinguishable. Here we briefly review the history of low-temperature chlorophyll fluorescence methods and the characteristics of the acquired emission spectra in oxygen-producing organisms. We discuss the contribution of different photosynthetic complexes and physiological processes to fluorescence emission at 77 K in cyanobacteria, green algae, heterokont algae, and plants. Furthermore, we describe practical aspects for obtaining and presenting 77 K fluorescence spectra.     

Spectral resolution of the split EPR signals induced by illumination at 5 K from the S1, S3, and S0 states in photosystem II

S-State-dependent split EPR signals that are induced by illumination at cryogenic temperatures (5 K) have been measured in spinach photosystem II without interference from the Y(D)* radical in the g approximately 2 region. This allows us to present the first decay-associated spectra for the split signals, which originate from the CaMn4 cluster in magnetic interaction with a nearby radical, presumably Y(Z)*. The three split EPR signals that were investigated, "Split S1", "Split S3", and Split S0", all exhibit spectral features at g approximately 2.0 together with surrounding characteristic peaks and troughs. From microwave relaxation studies we can reach conclusions about which parts of the complex spectra belong together. Our analysis strongly indicates that the wings and the middle part of the split spectrum are parts of the same signal, since their decay kinetics in the dark at 5 K and microwave relaxation behavior are indistinguishable. In addition, our decay-associated spectra indicate that the g approximately 2.0 part of the "Split S1" EPR spectrum contains a contribution from magnetically uncoupled Y(Z)* as judged from the g value and 22 G line width of the EPR signal. The g value, 2.0033-2.0040, suggests that the oxidation of Y(Z) at 5 K results in a partially protonated radical. Irrespective of the S state, a small amount of a carotenoid or chlorophyll radical was formed by the illumination. However, this had relaxation and decay characteristics that clearly distinguish this radical from the split signal spectra. In this paper, we present the "clean" spectra from the low-temperature illumination-induced split EPR signals from higher plants, which will provide the basis for further simulation studies.     

Vibrational dynamics and heat capacity of polyglycine I

Earlier works on polyglycine I suffer from several infirmities, such as the dynamic methylene group being replaced by a mass unit and the use of poorly resolved inelastic neutron spectra, which have resulted in wrong assignments and imprecise profile of dispersion curves. In addition, the density-of-states and heat capacity variation as a function of temperature are being reported for the first time. The heat capacity is in good agreement with the measurements reported earlier by Roles and Wunderlich within a certain range (230-350 K). Deviations set in beyond this could be due to the presence of two crystalline states (I and II) in the sample used for the heat capacity measurements.     

Changes in structure and stability of calbindin-D(28K) upon calcium binding

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.     

Physiological implications of K accumulation in heart muscle

K+-selective microelectrodes in conjugation with the voltage clamp technique were used to examine the voltage and time dependence of K+ efflux and accumulation in cardiac muscle. K+ efflux per action potential is about 10 to 30 pmoles/cm2 per sec. Accumulation of K+ in the paracellular space plays an important role in regulation of action potential duration, so that the [K+]o prior to generation of an action potential determines the duration of following action potential. This regulation is brought about by the shift of inward rectifying K+ current along the voltage axis, so at higher [K+]o there is more outward current at plateau potentials. Monitoring [K+]o after a period of rapid beating provides quantitative data regarding Na-pump activity. The data suggest the Na-pump is electrogenic, making it difficult to assess the extent of K+ accumulation from the measurements of resting potential alone. These studies indicate that changes in [K+]o not only reflect outward membrane currents and Na-pump activity, but also play an important physiological regulatory role in determining the duration of the action potential.     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

A new model for the K+-induced macromolecular structure of guanosine 5'-monophosphate in solution.

The (31)P NMR spectra of (TMA)(2)(5'-GMP), where TMA is [(CH(3))(4)N](+) and 5'-GMP is guanosine 5'-monophosphate, and K(2)(5'-GMP), containing various amounts of KCl or TMACl, have been obtained at 2 degrees C. Variable-temperature spectra have also been obtained for K(2)(5'-GMP). The TMA(+) ion serves to neutralize the charge on the dianionic 5'-GMP and permits the added K(+) to bond preferentially in structure-forming sites. (1)H NMR spectra (one- and two-dimensional) have been obtained for K(2)(5'-GMP) and used to assign the proton resonances in the self-associated structures and determine that all residues have the anti glycosidic conformation. The (31)P and (1)H NMR spectra are very complex and indicate the presence of a large number of molecular environments and a structural variation dependent upon the mole ratio of 5'-GMP to K(+). A new model for the solution structure is proposed in which the 5'-GMP forms a pseudo-four-stranded helix with guanine-guanine hydrogen bonding forming a continuous helical strand, rather than the usual planar G-tetrad structure. The guanine-guanine hydrogen bonding sites are the same as that found in a G-tetrad. The K(+) ions would be located in the center of the helix and bonding to the carbonyl oxygens. They are interacting with the phosphates as well. Integration data from the largest sized species give an estimate of 14.3 +/- 1.1 residues in a helical structure.     

The far-infrared properties of polyamino acids

The far-infrared absorption between 3 and 650 cm^?1 of several amino acids and polyamino acids is measured under a variety of conditions. The far-infrared spectra of amino acids and short-chain oligomers are extremely temperature dependent with the appearance of numerous narrow absorption lines at 4.2 K. These absorption lines broaden or disappear at 300 K or for absorption measurements on samples in solution. For longer polymers, relatively broad nearly temperature-independent absorption lines are observed with little difference between the spectra of polymers in solution or in the solid state. These results are explained in terms of the crystalline order of the samples.