Reactions of H2, CO, and O2 with active [NiFe]-hydrogenase from Allochromatium vinosum. A stopped-flow infrared study

The Ni-Fe site in the active membrane-bound [NiFe]-hydrogenase from Allochromatium vinosum can exist in three different redox states. In the most oxidized state (Ni(a)-S) the nickel is divalent. The most reduced state (Ni(a)-SR) likewise has Ni(2+), while the intermediate state (Ni(a)-C) has Ni(3+). The transitions between these states have been studied by stopped-flow Fourier transform infrared spectroscopy. It is inferred from the data that the Ni(a)-S --> Ni(a)-C* and Ni(a)-C* --> Ni(a)-SR transitions induced by dihydrogen require one of the [4Fe-4S] clusters to be oxidized. Enzyme in the Ni(a)-S* state with all of the iron-sulfur clusters reduced reacts with dihydrogen to form the Ni(a)-SR state in milliseconds. By contrast, when one of the cubane clusters is oxidized, the Ni(a)-S state reacts with dihydrogen to form the Ni(a)-C state with all of the iron-sulfur clusters reduced. The competition between dihydrogen and carbon monoxide for binding to the active site was dependent on the redox state of the nickel ion. Formation of the Ni(a)-S.CO state (Ni(2+)) by reacting CO with enzyme in the Ni(a)-SR and Ni(a)-S states (Ni(2+)) is considerably faster than its formation from enzyme in the Ni(a)-C* (Ni(3+)) state. Excess oxygen converted hydrogen-reduced enzyme to the inactive Ni(r)* state within 158 ms, suggesting a direct reaction at the Ni-Fe site. With lower O(2) concentrations the formation of intermediate states was observed. The results are discussed in the light of the present knowledge of the structure and mechanism of action of the A. vinosum enzyme.     

The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E-E* conformational change.

The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca2+ binds only to E and interior Ca2+ binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of cECa2, the enzyme with Ca2+ bound at the exterior site, was measured at different Ca2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca2+ by reaction with 0.5 mM [gamma-32P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO4, 40 mM Mops/KOH, pH 7.0, 25 degrees C). The sigmoidal dependence on free exterior calcium concentration of the concentration of cECa2, measured as [32P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K0.5 is 2.8 microM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca2+ to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca2+ is determined by the state of phosphorylation of the CaATPase.     

Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor?

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.     

Mechanism of superoxide and hydrogen peroxide formation by fumarate reductase,succinate dehydrogenase,and aspartate oxidase

Oxidative stress is created in aerobic organisms when molecular oxygen chemically oxidizes redox enzymes, forming superoxide (O2*-) and hydrogen peroxide (H2O2). Prior work identified several flavoenzymes from Escherichia coli that tend to autoxidize. Of these, fumarate reductase (Frd) is notable both for its high turnover number and for its production of substantial O2*- in addition to H2O2. We have sought to identify characteristics of Frd that predispose it to this behavior. The ability of excess succinate to block autoxidation and the inhibitory effect of lowering the flavin potential indicate that all detectable autoxidation occurs from its FAD site, rather than from iron-sulfur clusters or bound quinones. The flavin adenine dinucleotide (FAD) moiety of Frd is unusually solvent-exposed, as evidenced by its ability to bind sulfite, and this may make it more likely to react adventitiously with O2*-. The autoxidizing species is apparently fully reduced flavin rather than flavosemiquinone, since treatments that more fully reduce the enzyme do not slow its turnover number. They do, however, switch the major product from O2*- to H2O2. A similar effect is achieved by lowering the potential of the proximal [2Fe-2S] cluster. These data suggest that Frd releases O2*- into bulk solution if this cluster is available to sequester the semiquinone electron; otherwise, that electron is rapidly transferred to the nascent superoxide, and H2O2 is the product that leaves the active site. This model is supported by the behavior of "aspartate oxidase" (aspartate:fumarate oxidoreductase), an Frd homologue that lacks Fe-S clusters. Its dihydroflavin also reacts avidly with oxygen, and H2O2 is the predominant product. In contrast, succinate dehydrogenase, with high potential clusters, generates O2*- exclusively. The identities of enzyme autoxidation products are significant because O2*- and H2O2 damage cells in different ways.     

The oxygenated complexes of the two catalytically active oxidation-reduction states of L-tryptophan-2,3-dioxygenase.

The oxygenated complexes of the two catalytically active forms of pseudomonad and rat liver L-tryptophan-2,3-dioxygenase (EC 1.13.11.11) have been studied. As was previously reported (ISHIMURA, Y., NORZAKI, M., HAYAISHI, O., TAMURA, M., AND YAMAZAK-I I. (1970) J. Biol. Chem. 245, 3593-3602), we observe that the fully reduced form of pseudomonad tryptophan oxygenase during steady state catalysis exists predominantly as the L-tryptophan ferroheme-O2 enzyme complex (lambdamax = 415 nm, 540 nm, 570 nm). However, during steady state catalysis by a half-reduced form of both the pseudomonad and hepatic enzymes, the predominant species present manifest absorption spectra indicative of ternary complexes in which all the heme exists as ferriheme (Soret, 407 nm), there being no trace of a ferroheme-O2 complex. Carbon monoxide is a competitive inhibitor with respect to molecular oxygen of catalysis by either the half-reduced or fully reduced forms of pseudomonad tryptophan oxygenase. During steady state catalysis in the presence of CO, the fully reduced form of the enzyme exists as a mixture of the oxyferroheme (Soret = 415 nm) and carboxyferroheme (Soret = 421 nm) enzyme complexes. However, if the same experiment is repeated with the half-reduced form of the pseudomonad enzyme, all of the enzyme is in the ferriheme state, even though CO is inhibiting this form of the enzyme to the same degree as it does the fully reduced form. We conclude that for the half-reduced form of pseudomonad tryptophan oxygenase the substrate, O2, and the inhibitor, CO, are not binding to the heme moieties, but are bound elsewhere, presumably to the Cu(I) moieties. Examination of the kinetic mechanisms of the half-reduced and fully reduced forms of pseudomonad tryptophan oxygenase using the inhibitors carbon monoxide and 5-fluorotryptophan confirmed that the fully reduced enzyme binds L-tryptophan before O2 (FORMAN, H., AND FEIGELSON, P. (1971) Biochemistry 10, 760-763) and that for the half-reduced enzyme O2 binds first. In the presence of 5-fluorotryptophan a relatively stable oxyferroheme enzyme complex was generated with the fully reduced form of pseudomonad tryptophan oxygenase. Thus, saturation of the catalytic site alone either with the substrate, L-tryptophan, or the competitive inhibitor, 5-fluorotryptophan, enhances binding of O2 to the ferroheme moieties of the enzyme. The resistance of this complex to photolysis indicates that the bound molecular oxygen is predominantly present as superoxide, O2-minus.     

Formation of the covalent chymotrypsin.antichymotrypsin complex involves no large-scale movement of the enzyme

alpha(1)-Antichymotrypsin is a member of the serine proteinase inhibitor, or serpin, family that typically forms very long-lived, enzymatically inactive 1:1 complexes (denoted E*I*) with its target proteinases. Serpins share a conserved tertiary structure, in which an exposed region of amino acid residues (called the reactive center loop or RCL) acts as bait for a target proteinase. Within E*I*, the two proteins are linked covalently as a result of nucleophilic attack by Ser(195) of the serine proteinase on the P1 residue within the RCL of the serpin. This species is formally similar to the acyl enzyme species normally seen as an intermediate in serpin proteinase catalysis. However, its subsequent hydrolysis is extremely slow as a result of structural changes within the enzyme leading to distortion of the active site. There is at present an ongoing debate concerning the structure of the E*I* complex; in particular, as to whether the enzyme, bound to P1, maintains its original position at the top of the serpin molecule or instead translocates across the entire length of the serpin, with concomitant insertion of RCL residues P1-P14 within beta-sheet A and a large separation of the enzyme and RCL residue P1'. We report time-resolved fluorescence energy transfer and rapid mixing/quench studies that support the former model. Our results indicate that the distance between residue P1' in alpha(1)-antichymotrypsin and the amino terminus of chymotrypsin actually decreases on conversion of the encounter complex E.I to E*I*. These results led us to formulate a comprehensive mechanism that accounted both for our results and for those of others supporting the two different E*I* structures. In this mechanism, partial insertion of the RCL, with no large perturbation of the P1' enzyme distance, is followed by covalent acyl enzyme formation. Full insertion can subsequently take place, in a reversible fashion, with the position of equilibrium between the partially and fully inserted complexes depending on the particular serpin-proteinase pair under consideration.     

Effect of 17O2 and 13CO on EPR spectra of nickel in hydrogenase from Chromatium vinosum

Oxygen, either molecular oxygen or a reduction adduct, can tightly bind in the vicinity of the two forms of trivalent nickel occurring in hydrogenase from Chromatium vinosum, as evident from studies with 17O-enriched O2. This oxygen is not in the first coordination sphere of nickel. As has been reported earlier for hydrogenase from Desulfovibrio gigas (Fernandez, V.M., Hatchikian, A.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), also the relative activity of the C.vinosum enzyme correlates well with the presence of only one of the two Ni(III) forms in the oxidized preparation. These results make it less likely that a specific oxygenation of only one of the Ni(III) forms would be the reason for the reversible inactivation of nickel hydrogenases by oxygen. Reaction of H2-reduced enzyme with 13CO now demonstrated beyond doubt that: (i) One 13CO molecule is a direct ligand to nickel in axial position; and (ii) hydrogen binds at the same coordination site as CO. It can also be concluded that hydrogen is not bound as a hydride ion, but presumably as molecular hydrogen. A simple way to explain the EPR spectra from the 13CO-adduct of the enzyme is to assume a monovalent state for the nickel.     

Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome c oxidase.

Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO (a2+ CuA+ a3(2+)-CO CuB+) cytochrome c oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence (a3+ CuA2+ a3(2+)-CO CuB+) and the fully reduced CO complexes were also studied. A band near 785 nm (epsilon approximately 50 M-1cm-1) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale (t1/2 approximately 7 ms) as CO recombined with cytochrome a3(2+). This band, which is attributed to the unliganded five coordinate ferrous cytochrome a3(2+), has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at approximately 710 nm (epsilon approximately 80 M-1cm-1) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome a, appears to be affected by the ligation state at the cytochrome a3(2+) site.     

Biochemical and biophysical studies on cytochrome c oxidase. XIX. An EPR study of the photodissociation of carboxycytochrome c oxidase in the presence of azide

1. The photodissociation reaction of the cytochrome c oxidase-CO compound in the presence of azide was studied by EPR at 15°K. Addition of CO in the dark to cytochrome c oxidase, partially reduced (2 electrons per 4 metal ions) in the presence of azide brings about a decrease in intensity of the azide-induced low-spin heme signal at g = 2.9, 2.2 and 1.67 and an increase in intensity of both the low-spin heme signal at g = 3 and the copper signal at g = 2. Subsequent illumination with white light at room temperature of this sample causes an enhancement of the azide-induced signal at g = 2.9, and a decrease in intensity of both signals at g = 3 and g = 2. It is shown that these changes in the EPR spectrum are reversible.

2. These results demonstrate that upon photodissociation, CO is replaced by azide wheras upon incubation in the dark CO expels azide from its binding site in cytochrome c oxidase.

3. Concomitantly with the binding of CO and dissociation of the azide molecule, and vice versa, electron redistributions occur as inferred from the changes in the intensity of the copper signal at g = 2.

4. The results are explained in a model of cytochrome c oxidase with either a common binding site (cytochrome a₃)^* for CO and azide or in a model with anti-cooperative interaction between two different sites of binding.

5. Similar types of experiments with cyanide instead of azide show that cyanide is more firmly bound to partially reduced cytochrome c oxidase than CO and azide. The affinity of ligands for partially reduced enzyme decreases in the sequence: cyanide, CO (dark), azide and CO (illuminated).     

Proximal ligand motions in H93G myoglobin.

Resonance Raman spectroscopy has been used to observe changes in the iron-ligand stretching frequency in photoproduct spectra of the proximal cavity mutant of myoglobin H93G. The measurements compare the deoxy ferrous state of the heme iron in H93G(L), where L is an exogenous imidazole ligand bound in the proximal cavity, to the photolyzed intermediate of H93G(L)*CO at 8 ns. There are significant differences in the frequencies of the iron-ligand axial out-of-plane mode nu(Fe-L) in the photoproduct spectra depending on the nature of L for a series of methyl-substituted imidazoles. Further comparison was made with the proximal cavity mutant of myoglobin in the absence of exogenous ligand (H93G) and the photoproduct of the carbonmonoxy adduct of H93G (H93G-*CO). For this case, it has been shown that H2O is the axial (fifth) ligand to the heme iron in the deoxy form of H93G. The photoproduct of H93G-*CO is consistent with a transiently bound ligand proposed to be a histidine. The data presented here further substantiate the conclusion that a conformationally driven ligand switch exists in photolyzed H93G-*CO. The results suggest that ligand conformational changes in response to dynamic motions of the globin on the nanosecond and longer time scales are a general feature of the H93G proximal cavity mutant.     

Cell-free expression of visual arrestin. Truncation mutagenesis identifies multiple domains involved in rhodopsin interaction.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. To further characterize rhodopsin/arrestin interactions we have utilized a rabbit reticulocyte lysate translation system to synthesize bovine visual arrestin. The translated arrestin (404 amino acids) was demonstrated to be fully functional in terms of its ability to specifically recognize and bind to phosphorylated light-activated rhodopsin (P-Rh*). Competitive binding studies revealed that the in vitro synthesized arrestin and purified bovine visual arrestin had comparable affinities for P-Rh*. In an effort to assess the functional role of different regions of the arrestin molecule, two truncated arrestin mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes. In vitro translation of the transcribed truncated mRNAs resulted in the production of arrestins truncated from the carboxyl terminus. The ability of each of the mutant arrestins to bind to dark (Rh), light-activated (Rh*), dark phosphorylated (P-Rh), and light-activated phosphorylated rhodopsin were then compared. Arrestin lacking 39 carboxyl-terminal residues binds specifically not only to P-Rh* but also to Rh* and P-Rh. This suggests that the carboxyl-terminal domain of arrestin plays an important regulatory role in ensuring strict arrestin binding selectivity to P-Rh*. Arrestin that has only the first 191 amino-terminal residues predominately discriminates the phosphorylation state of the rhodopsin; however, it also retains some binding specificity for the activation state. These results suggest that the amino-terminal half of arrestin contains key rhodopsin recognition sites responsible for interaction with both the phosphorylated and light-activated forms of rhodopsin.     

N-carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea. Thermodynamics and kinetics of the spontaneous reaction.

N-carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3-+ CO32-) was found to be 7 M-1.s-1 (v1 = k1* ['MFR'] ['CO2']) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s-1 (v2 = k2* [carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/['MFR']['CO2'] was 0.6 M-1 at pH 7.0 corresponding to a free energy change DeltaG degrees ' of + 1.3 kJ.mol-1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3- in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3- which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N-carboxymethanofuran to N-formylmethanofuran. From the concentrations of 'CO2' and of 'MFR' determined by 1D-NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1 [MFR] [CO2]). The second order rate constant k1 was found to be approximately 1000 M-1 x s-1 at 300 K and pH values between 7.0 and 8. 0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.     

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.     

UV-A/blue-light inactivation of the 'metal-free' hydrogenase (Hmd) from methanogenic archaea.

H2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed 'metal-free' hydrogenase does not contain iron-sulfur clusters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron-sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component. Hmd does, however, harbour a low molecular mass cofactor of yet unknown structure. We report here that the iron found in Hmd is most probably functional after all. Further investigation was initiated by the discovery that Hmd is inactivated upon exposure to UV-A (320-400 nm) or blue-light (400-500 nm). Enzyme purified in the dark exhibited an absorption spectrum with a maximum at approximately 360 nm and which mirrored its sensitivity towards light. In UV-A/blue-light the enzyme was bleached. The cofactor extracted from active Hmd was also light sensitive. It showed an UV/visible spectrum similar to that of the active enzyme and was bleached upon exposure to light. Photobleached cofactor no longer had the ability to reconstitute active Hmd from the apoenzyme. When purified in the dark, Hmd consistently contained per monomer about one Fe, which was tightly bound to the cofactor. The iron was released from the enzyme and from the cofactor upon light inactivation. Hmd activity was inhibited by high concentrations of CO and CO protected the enzyme from light inactivation indicating that the iron in Hmd is of functional importance. Therefore, reference to Hmd as 'metal-free' hydrogenase is no longer appropriate.     

Carbon monoxide-dependent growth of Rhodospirillum rubrum.

Under dark, anaerobic conditions in the presence of sufficient nickel, Rhodospirillum rubrum grows with a doubling time of under 5 h by coupling the oxidation of CO to the reduction of H+ to H2. CO-dependent growth of R. rubrum UR294, bearing a kanamycin resistance cassette in cooC, depends on a medium nickel level ninefold higher than that required for optimal growth of coo+ strains.     

Reduction of oxygen by the electron transport chain of chloroplasts during assimilation of carbon dioxide.

In photosynthetically competent chloroplasts from spinach the quantum requirements for oxygen evolution during CO2 reduction were higher, by a factor often close to 1.5, than for oxygen evolution during reduction of phosphoglycerate. Mass spectrometer experiments performed under rate-limiting light indicated that an oxygen-reducing photoreaction was responsible for the consumption of extra quanta during carbon dioxide assimilation. Uptake of 18O2 during reduction of CO2 was considerably higher than could be accounted for by oxygen consumption during glycolate formation and by the Mehler reaction of broken chloroplasts which were present in the preparations of intact chloroplasts. The oxygen reducing reaction occurring during CO2 assimilation resulted in the formation of H2O2. This was indicated by a large stimulation of CO2 reduction by catalase, but not of phosphoglycerate reduction. Catalase could be replaced as a stimulant of photosynthesis by dithiothreitol or ascorbate, compounds known to react with superoxide radicals. There was no effect of dithiothreitol and ascorbate on phosphoglycerate reduction. A main effect of superoxide radicals and/or H2O2 was shown to be at the level of phosphoglycerate formation. Evidence for electron transport of oxygen was also obtained from 14CO2 experiments. The oxidation of dihydroxyacetonephosphate during a dark period or after addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone in the light was studied. The results indicated a link between the chloroplast pyridine nucleotide system and oxygen. Oxygen reduction during photosynthesis under conditions where light is rate limiting is seen as important in supplying the ATP which is needed for CO2 reduction but is not provided during electron transport to NADP. A mechanism is discussed which would permit proper distribution of electrons between CO2 and oxygen during photosynthesis.     

Picosecond time-resolved infrared spectroscopic investigation into electron localisation in the excited states of Re(i) polypyridyl complexes with bridging ligands.

Mono- and binuclear complexes of (Re(CO)3Cl) with dipyrido[2,3-a:3',2'-c]-6,7-dimethylphenazine (ppbMe2) were synthesised and their photophysical properties probed using picosecond time-resolved infrared spectroscopy (TRIR). Excitation of these complexes in solution at 400 nm produces short-lived excited states. The IR spectrum of the excited state of the mononuclear [Re(CO)3Cl(ppbMe2)] have nu(CO) bands shifted to higher wavenumber relative to those of the ground state. This is consistent with formation of a (3)MLCT excited state. The IR spectrum of the excited state of the bimetallic [(Re(CO)3Cl)2(micro-ppbMe2)] shows the formation of two distinct groups of nu(CO) bands. This is interpreted as the formation of two distinct Re sites arising from a localised MLCT state with formally oxidised Re centre and a formally reduced bridging ligand. The nu(CO) bands of the adjacent Re centre are affected by the reduction of the bridging ligand. On the IR timescale the excited state structure is best formulated as [Cl(CO)3Re(II)(micro-ppbMe2 *-)Re(I)(CO)3Cl].     

A simple method for calculating the dissociation constant of a receptor (or enzyme).unlabeled ligand complex from radioligand displacement measurements

A general procedure is described for determining the dissociation constant of a receptor (or enzyme).unlabeled ligand complex (EI) by analyzing the I-dependent displacement of bound radioligand (A*) from EA*. The procedure (which involves measuring free A* in the presence of I) requires a knowledge of the total concentrations of receptor ([E]t), unlabeled ligand ([I]t) and radioligand ([A*]t), and the dissociation constant of the EA* complex. The unknown Kd is obtained from five simple, sequential calculations which are valid for either high or low affinity competitive unlabeled ligands and are independent of total receptor concentration or initial degree of saturation with A*. The procedure also provides the information needed to construct a distribution curve of all enzyme and ligand species (E, EA*, EI, A*, I) as [I]t is varied.     

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.