PubMed · 5706623
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C Reuge. 1968. [Standardized method for hemoglobin determination].. https://doi.org/10.1016/s0035-2977(68)80080-x
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Cytochrome bd oxidase is a terminal bacterial oxidase containing three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be at a dinuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinone. One of the unique functional features of this enzyme is its resistance to high concentrations of cyanide (Ki in the millimolar range). With the appropriate selection of laser lines, the ligation and spin states of the b558, b595, and d hemes can be probed selectively by resonance Raman (rR) spectroscopy. Wavelengths between 400 and 500 nm predominantly excite the rR spectra of the b558 and b595 chromophores. Spectra obtained within this interval show a mixed population of spin and ligation states arising from b558 and b595, with the former more strongly enhanced at higher energy. Red excitation wavelengths (590-650 nm) generate rR spectra characteristic of chlorins, indicating the selective enhancement of the d heme. These rR results reveal that cytochrome bd oxidase "as isolated" contains the b558 heme in a six-coordinate low-spin ferric state, the b595 heme in a five-coordinate high-spin (5cHS) ferric state, and the d heme in a mixture of oxygenated (FeIIO2 <--> FeIIIO2-; d650) and ferryl-oxo (FeIV = O; d680) states. However, the rR spectra of these two chlorin species indicate that they are both in the 5cHS state, suggesting that the d heme is lacking a strongly coordinated sixth ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
The interaction of peroxidatic derivatives of cytochrome c oxidase with cyanide has been investigated by optical spectroscopy and the stopped-flow method. Two reactions were found in the conversion of peroxy cytochrome oxidase to its cyanide complex. The first reaction is characterized by the loss of the 607 nm band, an increase in absorbance at 655 nm, and a decrease in absorbance at 432 nm resulting from a blue-shift of the Soret band; this reaction occurred with a bimolecular rate constant of about 90 M-1 s-1. The second reaction is observed as an absorbance increase at 585 and 432 nm; the latter was due to a red-shift of the Soret band. This second process proceeded with a rate constant of about 22 M-1 s-1. Both reaction rates are linearly dependent on the concentration of cyanide between 5 and 100 mM. The reappearance of the 655 nm band at the completion of the first reaction suggests that cytochrome a3 becomes transiently high-spin, a finding which implies that cyanide is not initially bound to this heme center. It appears that preparations of oxidized CcO contain small but variable amounts of the peroxy form. The variable content of this form is probably responsible for the different response of oxidized oxidase to low concentrations of cyanide [Berka, V., Vygodina, T., Musatov, A., Nicholls, P., & Konstantinov, A. A. (1993) FEBS Lett. 315, 237-241] and may explain the biphasic reduction of the binuclear center with dithionite [Cooper, C. E., Junemann, S., Ioannidis, N., & Wrigglesworth, J. M. (1993) Biochim. Biophys. Acta 1144, 149-160].
The fluorescence-decay characteristics of the single tryptophan present in horseradish peroxidase (HRP) have been studied using dye-laser pulses and single-photon counting techniques. The decay was found to be dominated by a picosecond-lifetime component, with small contributions from two other lifetime components in the nanosecond range. The distance of the tryptophan residue was estimated from the fluorescence-energy transfer to the heme moiety using Förster's theory. The tryptophan residue was found to be approximately 1.2 nm from the heme moiety at neutral pH. Detailed analysis of the fluorescence-decay profiles using the maximum-entropy method (MEM) has been carried out. The results of the MEM analysis also showed a maximum amplitude peak at approximately 45 ps (at pH approximately 7) with a very small (< 5%) contribution from two other components. Similar results were obtained with the cyanide derivative of the enzyme (HRPCN) where the major lifetime component was found to be 58 ps at neutral pH. The picosecond component of fluorescence lifetimes of native HRP as well as of HRPCN were found to increase with decrease in pH in the range pH 6-3.5. Moreover, the native enzyme showed significant increase in the magnitude of this fast lifetime component at pH above 8. Such increase in the major lifetime component possibly indicated a conformational perturbation caused by pH change in the enzyme. However, the pH dependence of HRPCN, which is devoid of alkaline transition, showed that the shortest lifetime component remains almost unchanged over the pH range 6-11. This result showed that the alkaline transition in native HRP is associated with a structural change in the distal region of the heme center, which is absent in the cyanide-ligated enzyme. The results have been discussed with respect to understanding the pH-induced effects associated with salt bridge and hydrogen-bonding network in HRP.