Charge translocation coupled to electron injection into oxidized cytochrome c oxidase from Paracoccus denitrificans

Electrons were discretely injected into oxidized cytochrome c oxidase in liposomes by laser flash excitation of bound ruthenium [II] bispyridyl, and the membrane potential was recorded by time-resolved electrometry. Membrane potential is generated in a fast phase when an electron is transferred from the excited dye, via the CuA center, to heme a at a relative dielectric depth d inside the membrane [Zaslavsky, D., Kaulen, A. D., Smirnova, I. A., Vygodina, T., and Konstantinov, A. A. (1993) FEBS Lett. 336, 389-393]. Subsequently, membrane potential may develop further in a slower event, which is due to proton transfer into the enzyme from the opposite side of the membrane [Ruitenberg, M., Kannt, A., Bamberg, E., Ludwig, B., Michel, H., and Fendler, K. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4632-4636]. Here, we confirm that injection of the first electron into the fully oxidized cytochrome c oxidase from Paracoccus denitrificans is associated with a fast electrogenic 11 micros phase, but there is no further electrogenic phase up to 100 milliseconds when special care is taken to ensure that only fully oxidized enzyme is present initially. A slower electrogenic 135 micros phase only becomes apparent and grows in amplitude upon increasing the number of light flashes. This occurs in parallel with a decrease in amplitude of the 11 micros phase and correlates with the number of enzyme molecules that are already reduced by one electron before the flash. The electrogenic 135 micros phase does not appear with increasing flash number in the K354M mutant enzyme, where electron and proton transfer into the binuclear center is delayed. We conclude that the 135 micros phase, and its associated proton uptake, take place on electron injection into enzyme molecules where the binuclear heme a3-CuB site is already reduced by one electron, and that it is accompanied by oxidation of heme a with a similar time constant. Reduction of heme a is not associated with electrogenic proton uptake into the enzyme, neither in the fully oxidized nor in the one-electron-reduced enzyme. The extent of the electrogenic 135 micrcos phase also rules out the possibility that reduction of the binuclear center by the second electron would be coupled to proton translocation in addition to the electrogenic uptake of a proton.     

Electrogenic reactions of cytochrome bd

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O(2) and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H(2)O(2) with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O(2) and is apparently associated with full transmembrane translocation of approximately one charge.     

Gating current and potassium channels in the giant axon of the squid.

Gating current (Ig) underlying Na-channel activation is large enough to enable resolution of components both preceding and paralleling Na conductance (gNa) turn-on. For large depolarizations (beyond +20 mV), an additional "slow phase" of Ig is observed during a time when Na activation is already complete, but when K-channel opening is just becoming detectable. If Na- and K-channel gating are similar, the slow kinetics and long delay for K activation predict that K channel Ig must be relatively small and slow. Externally applied dibucaine almost totally blocks gNa and greatly reduces the fast (Na channel) Ig without altering gK or the Ig slow phase. The slow phase of Ig depends in part of the presence of functional K channels. Selective diminution in amplitude of the slow phase is consistently observed after a 30-min perfusion with both external and internal K-free media, a procedure which destroys nearly all K channels. This decrease of Ig amounts to approximately 10% of the total charge movements at +40 to +80 mV, with gating charge and K channels disappearing in a ratio of less than 1 e- per picosiemens of gK. These findings are consistent with the idea that part of the Ig slow phase represents gating current generated by the early steps in K-channel activation.     

Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.     

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.     

Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen

The reaction of cytochrome c oxidase with dioxygen has been studied by means of time-resolved measurements of electrical membrane potential (DeltaPsi). Microsecond time resolution was achieved by starting with the CO-inhibited enzyme, which was photolyzed after addition of oxygen. The time course of the reaction could be fitted by using a five-step sequential reaction as a model. The first two phases of the reaction, which correspond in time to binding of oxygen followed by formation of the P (peroxy) intermediate, as observed spectroscopically, are not associated with net charge displacement across the membrane. After this lag, DeltaPsi develops in three phases, which correspond in time to the conversion of P to the F (ferryl) intermediate, in a single phase, and conversion of F to O (the fully oxidized enzyme), in two phases. The amplitude of DeltaPsi was approximately equal for the P --> F and F --> O portions of the reaction. When the oxygen reaction is started with incompletely reduced enzyme, it will halt at the P or F state. When the reaction was allowed to proceed to the F state, but no further, only the fast phase of DeltaPsi formation was observed, whereas no DeltaPsi was generated if the reaction was halted at P. This finding places the assignments of phases in the electrometric data on a firmer basis-they are no longer based solely on temporal correspondence with phases in the spectroscopic data. To define the number of charges transferred across the membrane during the reaction, some kind of calibration is needed. For this purpose, another type of reaction-electron transfer following CO photolysis in the absence of oxygen ("backflow")-was studied. Parallel spectroscopic and electrometric measurements showed that the fast electron transfer from the low-spin heme to CuA in the backflow process results in approximately 11 times smaller amplitude of DeltaPsi as compared with DeltaPsi generated in the reaction of the reduced enzyme with oxygen (the polarity is also reversed). If it is assumed that transfer of an electron from the low-spin heme to CuA amounts to movement of a unit charge across half of the membrane dielectric, charge translocation in the reaction of the reduced enzyme with oxygen amounts to approximately 5.5 unit charges-the value predicted if all four protons pumped during the catalytic cycle are translocated during the oxidative part of the reaction.     

Transmembrane charge separation during the ferryl-oxo -> oxidized transition in a nonpumping mutant of cytochrome c oxidase

The N139D mutant of cytochrome c oxidase from Rhodobacter sphaeroides retains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002) Biochemistry 41, 13417-13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo --> oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Deltaphi in the ferryl-oxo --> oxidized transition begins with rapid vectorial electron transfer from CuA to heme a (tau approximately 15 micros), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (tau approximately 0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuB site and decelerates approximately 4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the "chemical" proton transferred from Glu-286 to the heme a3/CuB site. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the "pumped" proton from Glu-286 to the D-ring propionate of heme a3 or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (approximately 0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (approximately 1.5 ms).     

A redox study of the electron transport pathway responsible for generation of the slow electrochromic phase in chloroplasts

The amplitude of the slow phase of the electrochromic bandshift and the dark redox state of cytochrome b6, as well as its flash-induced turnover, have been measured as a function of ambient redox potential between +200 and -200 mV. Formation of a quinol-like donor with an Em,7 = +100 +/- 10 mV is required for generation of the slow phase. 80-100% of the amplitude of this signal with a t 1/2 = 3-4 ms is observed at -200 mV where cytochrome b6 was almost fully reduced (Em,7 of dark and flash-induced photoreduction was -30 mV and -75 mV, respectively). The change in the photoreduction of cytochrome b6 above 0 mV had an Em,7 of +50 mV, about 50 mV more negative than the midpoint at this pH for the onset of the slow electrochromic change. At potentials below -140 mV the amplitude of b6 photoreduction becomes small or negligible. The nature of the cytochrome b6 photoresponse is changed at potentials below -140 mV from a net photoreduction with a t1/2 = approximately less than 1 ms to a photooxidation with a t1/2 = 15-20 ms that is substantially slower than the electrochromic band-shift with a t1/2 = 3-4 ms. It is concluded that the slow electrochromic phase probably does not arise from a mechanism involving a turnover of cytochrome b6. From consideration of the possible flash-induced electron-transfer steps and alternative mechanisms for generation of the slow phase, it is suggested that it may arise from a redox-linked H+ pump involving the high potential iron-sulfur protein.     

The kinetics of the diffusion-controlled reaction of the binding of carbon monoxide to hemoglobin in glycerol-water mixtures of high viscosity

The kinetics of the binding of carbon monoxide to human hemoglobin and to ferrous horseradish peroxidase (HRP) have been studied by flash photolysis in mixtures of glycerol and water over a wide range of temperature and solvent viscosities. This was done in order that the influence of diffusion-control on the association rates could be determined. The binding of CO to HRP which is much slower than binding to Hb was devoid of diffusion effects. By contrast, the fast and slow phases of binding to Hb in the high viscosity solvents both displayed curved Arrhenius plots, consistent with a change from a chemical activationcontrolled process in the high temperature region to a diffusion-controlled process in the low temperature region. Analyses of the curved Arrhenius plots indicated that in the low temperature diffusion-controlled region, the activation enthalpy is similar to the activation energy of viscosity of the solvent, as might be expected for a diffusion-controlled reaction.Curve fitting of rate-temperature-viscosity data, assuming simultaneous chemical activation and diffusion-control, yielded factors by which the diffusion rate constants differ from that for reaction between uniformly reactive spheres of equal radii. For the fast Hb reaction, observed upon partial photolysis, this factor varies from 0.02 to 1.1, depending upon the solvent composition. For the slow Hb reaction, observed upon higher degrees of photolysis, this factor was 0.03 and 0.04. These factors were rationalized in terms of fractional surface reactivities and of a maximum allowable solid angle of entry of reactant to the binding site. It was concluded that the steric hindrance of T-state Hb (slow reaction) is much greater than R-state Hb (fast reaction).     

Proton-coupled electron equilibrium in soluble and membrane-bound cytochrome c oxidase from Paracoccus denitrificans

The pH dependence of electron and proton re-equilibration upon CO photolysis from two-electron-reduced aa3 oxidase was followed by time-resolved electrometry and optical spectroscopy. Optical spectroscopy on soluble Paracoccus denitrificans enzyme at alkaline pH revealed a slow (1 ms) component of electron re-equilibration coupled to the release of protons from the catalytic site. In the work [Br?ndén, M., et al. (2003) Biochemistry 42, 13178-13184], it was proposed that this proton is released from a water molecule in the catalytic site, located deep in the membrane dielectric. Movement of charged particles such as protons across the dielectric should create an electric potential. However, recording of the time course of the potential generation did not show any potential development in the millisecond time domain, but instead, potential generation was found with an apparent time constant of 50-100 micros. This potential was generated upon proton release from the level of the binuclear catalytic site through the K-channel, because mutation in this channel abolishes the potential generation altogether. The apparent inconsistency between results from optical spectroscopy and electrometry was solved by optical experiments on the membrane-incorporated enzyme. Reconstituting the enzyme into proteoliposomes speeds up the slow electron redistribution process by a factor of 10 and shows the same time constant as potential generation. The possible mechanism of such dramatic change in the rate of proton transfer is discussed.     

Internal electron transfer in cytochrome c oxidase: evidence for a rapid equilibrium between cytochrome a and the bimetallic site.

Internal electron-transfer reactions in cytochrome oxidase following flash photolysis of the CO compounds of the enzyme reduced to different degrees (2-4 electron equiv) have been followed at 445, 605, and 830 nm. Apart from CO dissociation and recombination, two kinetic phases are seen both at 445 and at 605 nm with rate constants of 2 x 10(5) and 1.3 x 10(4) s-1, respectively; at 605 nm, an additional phase with a rate constant of 400 s-1 is resolved. At 830 nm, only the second reaction phase (rate constant of 1.3 x 10(4) s-1) is observed. The amplitude of the first phase is largest with the two-electron-reduced enzyme, whereas that of the second phase is maximal at the three-electron-reduction level. Neither phase shows any marked pH dependence. The reaction in the first phase has a free energy of activation of 41 kJ mol-1 and an entropy of activation of -14 JK-1 mol-1. Analysis suggests that the two rapid reaction phases represent internal electron redistributions between the bimetallic site and cytochrome a, and between cytochrome a and CuA, respectively. The slow phase (400 s-1) probably involves a structural rearrangement.     

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K⁺ so that the membrane voltage approaches the Nernst potential for K⁺. With constant external K⁺ concentration, the time course of internal K⁺ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K⁺ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.     

Chromous ion reduction of mammalian cytochrome oxidase and some of its derivatives.

The reduction of cytochrome c oxidase by Cr2+, followed by means of stopped-flow spectrophotometry, exhibits two phases: the faster Cr2+-concentration-dependent reaction has an initial rate constant of 1.1 X 10(4)M-1-S-1, but reaches a rate limit at high concentration of reductant; the slower phase is concentration-independent with a rate of 0.3S-1. The activation energies of the fast and the slow processes are 35 and 71 kJ/mol respectively. The reduction kinetics of the mixed-valence CO complex and the cyanide-inhibited enzyme were compared with those of the fully oxidized forms: both the liganded species have a fast phase identical with that found in the oxidized oxidase. A comparison of the kinetic difference spectra obtained for the fast phase of reduction of oxidized oxidase with those obtained on reduction of the liganded species suggests that the rapid phase arises from the reduction ofhaem a, and the slow phase from the reduction of haem a3.     

Kinetics of carbon monoxide and oxygen binding to hemoglobin in human red blood cell suspensions studied by laser flash photolysis

The reaction kinetics of the binding of CO and O₂ to hemoglobin (Hb) in human red blood cell (RBC) suspensions have been examined using a 300 ns dye laser to photodissociate HbCO or HbO₂. Fast (halftime1?0 μs) and slow (5?ms) processes were seen after photolysis. The results indicate that neither the rate constants nor the activation energies for the binding of CO to the fast reacting form of Hb in the RBC are significantly different from that measured in solution in spite of the different environments. Rate constants determined for O₂ binding in RBC were intermediate between rates observed for reaction with fast and slow reacting forms of Hb and probably consist of contributions from each. The slow recombination of CO and O₂ probably has contributions both from reaction with slow reacting forms of Hb and from ligand that had diffused away from the RBC after photolysis.     

Evidence for an electrogenic and a non-electrogenic component in the slow phase of the P515 response in chloroplasts

The flash-induced P515 absorbance change in intact chloroplasts consists of a fast and a slow phase. There is disagreement in the literature over the origin of the slow phase. Here we argue that the flash-induced slow phase in P515 absorbance change is composed of two different components. One component is most probably due to the electrogenic Q-cycle associated with the cytochrome b/f complex. The second component has decay kinetics that are much slower than the electrogenic reactions. We suggest that the second component is due to a non-electrogenic reaction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DQH₂ durohydroquinone - MV methylviologen - P515 Absorbance change at 518 nm     

Turnover and short-term regulation of fatty acid binding protein in liver

Rat hepatic fatty acid binding protein (hFABP) may play an important role in the intracellular transport and metabolism of fatty acids. Recent reports have suggested a substantial circadian variation in the amount of hFABP in liver, and a half-life of less than 2 h for this protein has been inferred. In the present study, the kinetics of hFABP turnover were examined directly. hFABP half-life measured after pulse labeling with NaH14CO3 was 3.1 days compared with 2.9 days for total cytosol protein. Following double-isotope labeling, the charge isoforms of hFABP showed similar rates of turnover, all of which were slower relative to whole cytosol protein turnover. Following a 48-h fast, total liver hFABP measured by immunoassay fell 65%, paralleling a 60% fall in total cytosol protein. Refeeding for 24 h did not lead to a significant recovery of either hFABP or total cytosol protein content. No significant change was observed in hFABP abundance between mid-light and mid-dark periods of a 24-h light-dark cycle. These studies showed that hFABP has a relatively slow rate of turnover and that it is not acutely modulated by dietary or diurnal influences.     

Glucose modulates [Ca2+]i oscillations in pancreatic islets via ionic and glycolytic mechanisms

Pancreatic islets of Langerhans display complex intracellular calcium changes in response to glucose that include fast (seconds), slow ( approximately 5 min), and mixed fast/slow oscillations; the slow and mixed oscillations are likely responsible for the pulses of plasma insulin observed in vivo. To better understand the mechanisms underlying these diverse patterns, we systematically analyzed the effects of glucose on period, amplitude, and plateau fraction (the fraction of time spent in the active phase) of the various regimes of calcium oscillations. We found that in both fast and slow islets, increasing glucose had limited effects on amplitude and period, but increased plateau fraction. In some islets, however, glucose caused a major shift in the amplitude and period of oscillations, which we attribute to a conversion between ionic and glycolytic modes (i.e., regime change). Raising glucose increased the plateau fraction equally in fast, slow, and regime-changing islets. A mathematical model of the pancreatic islet consisting of an ionic subsystem interacting with a slower metabolic oscillatory subsystem can account for these complex islet calcium oscillations by modifying the relative contributions of oscillatory metabolism and oscillatory ionic mechanisms to electrical activity, with coupling occurring via K(ATP) channels.     

Kinetics of CO and O(2) binding to human serum albumin-heme hybrid

The kinetics of the CO and O(2) binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-?N-(2-methylimidazolyl)?octanoyloxymethyl]-5,10,15, 20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [(fast), (slow)] were determined to be 4.9 x 10(6) M(-)(1) s(-)(1) and 6.7 x 10(5) M(-)(1) s(-)(1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O(2) photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG() of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.     

The relation of ligand binding to redox state in the hexa-heme nitrite reductase of Wolinella succinogenes.

Ligand binding reactions and the relation between redox state and ligand binding in the hexa-heme nitrite reductase of Wolinella succinogenes have been studied using laser flash photolysis. On a picosecond time scale, a rapid excursion was observed corresponding to the breaking and reforming of an iron histidine bond. With the CO derivative, a geminate reaction was observed with a rate of 3 ns-1. On a nanosecond time scale, no slower geminate reactions were observed. For the cyanide derivative, no geminate reactions were observed at either time scale. The second order reaction of CO with the enzyme had a time course consisting of two distinct components. This time course changed in form as the enzyme came to equilibrium with CO, and the slower rebinding component was replaced by a faster rebinding component. It is suggested that CO binding enhances reduction of a heme with an unusually low redox potential and opens the structure of the active site to allow a faster second order reaction of CO. The proportion of the geminate CO reaction was unchanged, consistent with changes relatively remote from the ligand binding site. The second order reactions of cyanide also showed that redox effects influence its rebinding reaction. Adding cyanide to the CO complex of nitrite reductase showed that the two ligands have distinct heme binding sites.     

The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes

The aim of this work was to characterize the effects that two commonly used "caged" calcium buffers (NP-EGTA and nitr-5) have on the amplitude and time course of decay of the calcium transient. We made quantitative measurements of both free and total calcium using the measured buffering properties of the cell. Intracellular calcium concentration ([Ca(2+)](i)) was measured with fluo-3 in rat ventricular myocytes. Incorporation of the buffer NP-EGTA decreased both the amplitude and rate of decay of the caffeine response. The slowing could be quantitatively accounted for by the measured increased buffering. These effects were removed by photolysis of NP-EGTA. Similar results were obtained with nitr-5 except that the effects were not completely removed by photolysis. This was shown to be due to the persistence of a component of the increased buffering after photolysis. Both buffers decreased the amplitude of the systolic calcium transient. However, although nitr-5 produced a simple slowing of the decay, NP-EGTA resulted in an initial rapid phase of decay. This rapid phase of decay is attributed to calcium binding to NP-EGTA. This work represents the first quantitative analysis of the effects that extra buffering by a fast and a slow calcium chelator may have on the calcium transient.