Near-infrared spectra of Scapharca homodimeric hemoglobin: characterization of the deoxy and photodissociated derivatives.

The near-infrared charge transfer band at 760 nm (band III) has been investigated in deoxy and photodissociated dimeric Scapharca hemoglobin. At 300 K, the 10-ns spectrum of the carbonmonoxy derivative photoproduct is shifted by about 6 nm toward longer wavelengths with respect to the deoxy spectrum, both in buffer and in glycerol/buffer solutions. Moreover, the band III peak occurs at about the same wavelength at 300 K and at 10 K for the 10-ns photodissociated derivative, whereas in the deoxy derivative large changes in peak position and linewidth are observed as a function of temperature. These findings suggest that in dimeric Scapharca hemoglobin the photoproduct has not relaxed after 10 ns. The complete time dependence of the relaxation process has been studied both in buffer and in glycerol/buffer solutions at room temperature. The relaxation from the photoproduct to the deoxy species occurs on a microsecond time scale, in line with recent optical absorption and resonance Raman measurements.     

Glass transition of deoxymyoglobin probed by optical absorption spectroscopy

We have measured the absorption spectrum of horse deoxymyoglobin in glycerol-water mixture around 430 nm in the 130 - 320 K temperature range. The observed asymmetric spectral shape of the Soret band was analyzed using a configuration-coordinate model. The results support the idea that myoglobin is liquid-like at physiological temperatures, but is glass-like below about 250 K. The equilibrium position of the iron atom in the heme group in the electronic excited state was estimated from the determined parameter values.     

Tryptophan-heme pi-electrostatic interactions in cytochrome f of oxygenic photosynthesis

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.     

Electron transfer process in cytochrome oxidase after pulse radiolysis

The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis. With the decay of the MNA radical, the absorption at 445 and 605 nm, a characteristic to ferrous heme a of the oxidase, increased. The kinetic difference spectrum obtained was similar to that of the fully reduced minus the fully oxidized form of the oxidase, and was not different from that obtained in the reaction of the MNA radical with the mixed valence CO complex of the oxidase, where heme a3 is the CO-bound reduced form with heme a oxidized. This suggests that the absorption changes at 445 and 605 nm arise from the reduction of heme a, not heme a3. In order to elucidate the contribution of "visible" copper in this reaction, the absorption of the oxidase in the near-infrared region was measured. A decrease of the 830 nm band due to the reduction of visible copper was detected with a half-life of 5 microseconds. This absorption change obeyed pseudo-first order kinetics and its rate constant increased with the concentration of the oxidase. This suggests that the absorption change at 830 nm is followed by a bimolecular reaction of the MNA radical with visible copper of the oxidase. After the first phase of the reduction, the return of the 830 nm band corresponding to oxidation of the copper was observed with a half-life of 100 microseconds. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme a increased. The rates of oxidation of the copper were identical to those of the reduction of heme a and independent of the oxidase concentration. This suggests that the MNA radical reacts with visible copper of the oxidase with a second order rate constant of 1.5 X 10(9) m-1 s-1 and subsequently the electron flows to heme a by intramolecular electron migration with a first order rate constant of 1.8 X 10(4) s-1. An activation energy of the intramolecular electron transfer was calculated to be 2.8 kcal/mol in the range 4-33 degrees C.     

Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation

The protein folding character of cyt c was studied with the use of a photocleavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 cyt c, the Soret absorption band slightly blue shifted compared with the unlabeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligation to the heme iron disappeared, and its resonance Raman spectrum was characteristic of a six-coordinate low-spin species, all characters demonstrating coordination of a non-native ligand, probably a histidine, instead of Met80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c was altered, and the transition midpoint concentration value of guanidine hydrochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the modification, which showed perturbation of the structure and decrease in protein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 nm absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordination and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorbance with a rate constant of 21 000 +/- 4000 s(-)(1) during refolding of cyt c initiated by irradiation of a 308 nm pulse on the NBz-Met65 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with that reported by the tryptophan fluorescence study [Shastry, M. C. R. S., and Roder, H. (1998) Nat. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate constant of 13 +/- 3 s(-)(1), and was not seen in the absence of GdnHCl.     

Nature of the principal photointermediate of halorhodopsin

Two alternative hypotheses have been presented as to the nature of the principal halorhodopsin photointermediate: a) it is a form whose its absorption band is shifted from the 575 nm position to 500 or 520 nm, and b) it is a form whose absorption band is shifted to only about 565 nm, but with an altered band shape so it exhibits a fortuitous difference peak near 500 nm. Such a shift with a maximum near 500 nm is also obtained in the dark when chloride is removed from the sample, suggesting the hypothesis that the spectral changes reflect the transient detachment of chloride from a binding site (Ogurusu et al, J. Biochem. Tokyo 95, 1073-1082, 1984). Comparison of the quantum yields of flash-induced absorption changes in halorhodopsin and bacteriorhodopsin strongly suggests, however, that hypothesis b) is untenable.     

Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.     

Proton interactions in the resting form of cytochrome oxidase.

The effect of pH on the near-UV absorption spectrum of cytochrome oxidase has been examined. Several lines of evidence implicate a proton binding site that can modulate the optical properties of cytochrome alpha 3 in the resting enzyme. Changing the pH within the range 6.5-10.5 was found to reversibly shift the position of the Soret band over an 11-nm range. The lower pH values caused a progressive blue shift in the Soret band, whereas the high-pH range promoted a gradual red shift. Limiting band positions were approximately 416 and 427 nm. The incubation time required to reach a stable band position varied somewhat as did the actual extent of the shift. In most cases, the shift was associated with an isosbestic point. A pH titration profile for the apparent equilibrium position of the Soret band was obtained. Nonlinear least-squares fitting to a scatter plot, assuming a single acid/base group, showed an apparent pKa of 7.8. Magnetic circular dichroism (MCD) spectra of the low-pH form at 416 nm, the high-pH form at 427 nm, and the cyanide derivative at 428 nm were compared. No evidence of a high-pH-dependent low-spin transition or a change in the redox state of cytochrome a3 was found, confirming earlier work [Baker, G. M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Subtraction of ferricytochrome a [spectrum taken from Vanneste, W. H. (1966) Biochemistry 5, 838-848] from a series of blue-shifting spectra showed a band at 414 nm that progressively gained amplitude and a band at 430 nm that correspondingly lost amplitude. A series of red-shifting spectra showed the opposite behavior with a clear isosbestic point being evident in both cases. The difference extinction change at 414 and 430 nm depended linearly on the position of the Soret band, both showing a reversible dependence on pH. The 430-nm band is noted to be unusually red-shifted for high-spin ferric heme a. An additional, pH-insensitive band was observed at 408-410 nm which was eliminated by treatment with cyanide. The kinetics of the pH-induced blue shift and red shift were obtained at 416 nm by using dual-wavelength method and found to be biphasic, despite the occurrence of an isosbestic point.(ABSTRACT TRUNCATED AT 400 WORDS)     

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4 degrees C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP (MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met-Lys-Thr (MKT) at the N-terminus, CCP (MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP (MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP (MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP (MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (< or = 5%) in the spectrum of CCP (MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (lambda CT = 637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP (MKT) (lambda CT = 637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Interaction of bd-type quinol oxidase from Escherichia coli and carbon monoxide: Heme d binds CO with high affinity

Comparative studies on the interaction of the membrane-bound and detergent-solubilized forms of the enzyme in the fully reduced state with carbon monoxide at room temperature have been carried out. CO brings about a bathochromic shift of the heme d band with a maximum at 644 nm and a minimum at 624 nm, and a peak at 540 nm. In the Soret band, CO binding to cytochrome bd results in absorption decrease and minima at 430 and 445 nm. Absorption perturbations in the Soret band and at 540 nm occur in parallel with the changes at 630 nm and reach saturation at 3-5 microM CO. The peak at 540 nm is probably either beta-band of the heme d-CO complex or part of its split alpha-band. In both forms of cytochrome bd, CO reacts predominantly with heme d. Addition of high CO concentrations to the solubilized cytochrome bd results in additional spectral changes in the gamma-band attributable to the reaction of the ligand with 10-15% of low-spin heme b558. High-spin heme b595 does not bind CO even at high concentrations of the ligand. The apparent dissociation constant values for the heme d-CO complex of the membrane-bound and detergent-solubilized forms of the fully reduced enzyme are about 70 and 80 nM, respectively.     

Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome c oxidase from bovine heart

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca²⁺ corresponds actually to a first derivative (differential) of the heme a ²⁺ absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a ²⁺ a ₃ ³⁺ -CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced heme a in the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca²⁺. The spectrum of heme a ²⁺ in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ～10 nm (589 cm^?1) corresponding to the perpendicularly oriented electronic π→π* transitions B _0x and B _0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of heme a ²⁺ originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838–848). The differences indicate that the overall γ-band of heme a ²⁺ in cytochrome oxidase contains in addition to the B _0x and B _0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of heme a ²⁺ contains significant contribution from heme a ₃ ²⁺ . The reconstructed absorption band of heme a ²⁺ in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm^?1) corresponds most likely to the individual Q _0y transition of heme a, whereas the Q _0x transition contributes only weakly to the spectrum.     

Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy

The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L). The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H). In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.     

高粱叶片磷酸烯醇式丙酮酸羧化酶溶液构象状态的紫外差示吸收光谱研究

PEP诱导产生的差光谱在237nm是一强负峰,在252nm附近呈宽负峰。Mg~(2+)产生的差光谱在275nm附近为正的阔峰,在237nm处为一负峰。PEP、Mg~(2+)共同与酶作用的差光谱在263nm附近呈宽的负峰。正效应剂G6P、Gly及GG分别存在条件下PEP羧化酶的差光谱亦各具明显差异,在270nm以下光区内尤其显著。在284nm和291nm为两个负峰,Gly诱导的峰强度大于G6P的,而GG复合效应剂对此两峰的影响表现很大的协同作用。Mal作用于酶的差光谱在246nm处有一负峰。     

Purification and characterization of a catalase-peroxidase from the fungus Septoria tritici.

Three classes of heme proteins, commonly designated hydroperoxidases, are involved in the metabolism of hydrogen peroxide: catalases, peroxidases, and catalase-peroxidases. While catalases and peroxidases are widely spread in animals, plants, and microorganisms, catalase-peroxidases were characterized only in prokaryotes. We report here, for the first time, on a catalase-peroxidase in a eukaryotic organism. The enzyme was purified from the fungal wheat pathogen Septoria tritici, and is one of three different hydroperoxidases synthesized by this organism. The S. tritici catalase-peroxidase, designated StCP, is similar to the enzymes previously isolated from the bacteria Rhodobacter capsulatus, Escherichia coli, and Klebsiella pneumoniae, although it is significantly more sensitive to denaturing conditions. In addition to its catalatic activity StCP catalyzes peroxidatic activity with o-dianisidine, diaminobenzidine, pyrogallol, NADH, and NADPH as electron donors. The enzyme is a tetramer with identical subunits of 61,000 Da molecular weight. StCP shows a typical high-spin ferric heme spectrum with a Soret band at 405 nm and a peak at 632 nm, and binding of cyanide causes a shift of the Soret band to 421 nm, the appearance of a peak at 537 nm, and abolition of the peak at 632 nm. Reduction with dithionite results in a decrease in the intensity of the Soret band and its shift to 436 nm, and in the appearance of a peak at 552 nm. The pH optimum is 6-6.5 and 5.4 for the catalatic and peroxidatic activities, respectively. Fifty percent of the apparent maximal activity is reached at 3.4 mM and 0.26 mM for the catalatic and peroxidatic activities, respectively. The enzyme is inactivated by ethanol/chloroform, and is inhibited by KCN and NaN3, but not by the typical catalase inhibitor 3-amino-1,2,4-triazole.     

Photoacoustic study of iron-deficiency anaemia

A photoacoustic spectrum of a smear of whole blood clearly shows all of the details of the haem absorption in an optical spectrum and good optical absorption data without a data extraction procedure. There are three peaks; γ (420 nm), β (550 nm), and α (585 nm) in the band 350–700 nm. For iron-deficiency anaemia the peak/peak ratios, ratios of the value of peak γ to that of the peak β and peak α, and the integral/integral ratios, ratios of the value of the integral under the peak γ to that under the peak β, peak α, peak β and peak α are closely related to the variation of haemoglobin concentration. Linear relationships among the ratios and the Hb concentration are found. These ratios could be a good index of haem synthesis disturbance.     

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.     

Fast reactions in carbon monoxide binding to heme proteins.

Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.     

Charge-transfer complexes of plastoquinone and alpha-tocopherol quinone in vitro

It has been found that plastoquinone (PQ) and alpha-tocopherol quinone (alpha-TQ) can form quinhydrone-type charge-transfer complexes on PQH2 and alpha-TQH2, respectively, both in the crystalline state and in solutions of organic solvents. The charge-transfer spectra of PQ/PQH2 mixtures in hydrophobic solvents showed two bands: one at 349-358 nm, the other at 430-440 nm, one charge-transfer band at 351-355 nm occurring in water-miscible solvents. The intensity ratio of these two bands varied with changing PQ/PQH2 ratio. The charge-transfer spectra of alpha-TQ/alpha-TQH2 mixtures in all solvents investigated showed one peak at 361-367 nm and a broad shoulder within the range 400-540 nm, whose shape varied depending on the solvent used. In the infrared spectrum of PQ and alpha-TQ (1700-1600 cm(-1)) splitting of the carbonyl band occurred and was caused by the presence of two peak. In the spectra of quinhydrones the splitting disappeared, this being brought about by the appearance of a new peak at the position of splitting, which originated from the complexed quinones. The possibility of the formation of such complexes in thylakoid membranes is discussed.