Charles R. Hartzell

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

Abstract We report the resonance Raman spectra of cytochrome c oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome c , the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome c oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome c oxidase. There are, however, definite differences between spectra of solubilized cytochrome c oxidase and electron transport particles in the oxidized states.

The valence of copper and the role of superoxide in the D-galactose oxidase catalyzed reaction

Abstract Evidence for the involvement of Cu(III) in an enzymic reaction (that catalyzed by D-galactose oxidase) is reported. Superoxide dismutase inhibits the rate of the D-galactose oxidase catalyzed reaction and causes a small increase in the EPR signal due to Cu(II). Both ferricyanide and superoxide activate the enzyme (frequently 4 fold or greater) and cause a decrease (to essentially zero in some cases) in the intensity of the EPR signal. These and other results suggest that in its catalytic cycle the enzyme oscillates between Cu(I) and Cu(III) with superoxide bound to a Cu(II) state being only a fleeting intermediate. The Cu(III) enzyme is apparently the oxidant which converts the primary alcohol function of galactose to an aldehyde.

Indirect electrochemical titration of beef heart cytochrome c oxidase

Titration of beef heart cytochrome c oxidase with electrochemically generated reductant, methyl viologen cation radical MV+, and oxidant, molecular oxygen, has enabled cycling of the oxidase repeatedly from its totally oxidized to its totally reduced forms. Spectrocoulometric results clearly show that cytochrome oxidase accepts four electrons during both reduction with MV+ and oxidation with O2. Oxidation with coulometrically generated O2 produces oxidized oxidase, with no evidence of the “oxygenated” form.

Charge distribution in electron transport components: Cytochrome c and cytochrome c oxidase mixtures

Abstract Mixtures of cytochrome c oxidase and cytochrome c have been titrated by coulometrically generated reductant, methyl viologen radical cation, and physiological oxidant, O2. Charge distribution among the heme components in mixtures of these two redox enzymes has been evaluated by monitoring the absorbance changes at 605 and 550 nm. Differences in the pathway of the electron transfer process during a reduction cycle as compared to an oxidation cycle are indicated by variations found in the absorbance behavior of the heme components during successive reductive and oxidative titrations. It is apparent that the potential of the cytochrome a heme is dependent upon whether oxidation or reduction is occurring.

Evaluation of the energetics of cytochrome C oxidase in the absence of cytochrome C

E”’ = 340-350 mV and E”’ ‘ = 210 mV. Spe~~~p%tometric data alone werehLyldako determine the potential differences between cytochrome c and heme a L, and cytochrome c and heme aH. The heme aL and heme aH potentials were then set with respect to the cytochrome c potential, which was determined independently [2] . This method eliminated the need for potentiometric mediators which have varied effects on the cytochrome c oxidase [3,4]. Muijsers et al. [S] reported the potentials of the hemes of cytochrome c oxidase in the presence of cytochrome c (Eorheme a3 = 33.5-360 mV and Eofheme a = 200-2.50 mv), and asserted that the hemes titrated as indistinguishable entities in the absence of cytochrome c (.!?‘heme aa = 280 mV). Potentiometric mediators were preient in solution. Thus Muijsers et al. suggested that the hemes of cytochrome c oxidase are equivalent in purified cytochrome c oxidase and become split in the presence of ligands such as cytochrome c [5]. Wharton and Cusanovich [3] presented potentiometric data for purified cytochrome c oxidase in the presence of varying amounts of ferricyanide ranging

Reversible redox titrations of cytochrome c and cytochrome c oxidase using detergent solubilized electrochemically generated mediator-titrants.

Abstract The repetitive, reversible equilibrium redox cycling of cytochrome c , cytochrome c oxidase, or mixtures thereof has been made possible by the use of the oxidant, ferricinium ion. This ion is electrochemically generated by the use of non-ionic detergent solubilized ferrocene which is apparently incorporated as micelles and readily electron transfers with an electrode. The ferricinium-ferrocene couple equilibrates rapidly with these heme proteins. Electrochemically generated benzylviologen radical cations are used as the reductant. The EO′ values for cytochrome c oxidase at pH 7.0 are 209 ± 15 mv (2e−) and 340 ± 15 mv (2e−).

Spectroscopic studies on the copper(II) complexes of carnosine

Abstract Carnosine complexes with copper(II) ions were studied with magnetic resonance techniques over a wide range of ligand to metal ratios at various pH values. Water proton relaxation rates increased with decreasing carnosine to copper ratios until a molar ratio of 48 was reached. Over the ratio range of 48–2 carnosine molecules per copper ion, the relaxation rate decreased so that in the 2:1 carnosine-copper(II) complex, the water-copper(II) distance was estimated to be 1.92 A. Proton NMR studies revealed the broadening of imidazole proton lines at high mole ratios followed by other histidyl protons as the ratio decreased. The β-alanyl methylene protons were the last to be broadened by the addition of copper(II) ions. Carbon to copper(II) distances were determined for the carnosine to copper mole ratios of 500:1 and 5000:1. EPR spectra obtained at 93°K revealed the probable existence of four carnosine imidazoles as the sole coordinated ligands to copper(II) at high dipeptide-to-metal ratios (>10). At mole ratios below four, nuclear hyperfine lines characteristic of both monomeric and dimeric carnosine-copper(II) forms were observed. These results reveal that imidazole from carnosine is the sole ligand contributed to copper(II) for coordination over the pH range 5 to 7 at high carnosine to copper(II) ratios

Copper(II) complexes of carnosine, glycylglycine, and glycylglycine-imidazole mixtures

Abstract A comparative spectroscopic study of the copper(II) complexes of carnosine, glycylglycine and glycylglycine-imidazole mixtures was undertaken to gain a better understanding of the solution structure of copper(II)-carnosine. Potentiometric titration data were obtained and correlated with the visible absorption spectra, water proton relaxation rates, and low temperature electron paramagnetic resonance spectra. Changes in the visible spectra and water relaxation rates with increasing pH reflect the displacement of solvent oxygen ligands from the coordination sphere of copper(II) by the ligating groups found on the dipeptides. Estimates of the number of solvent molecules in rapid exchange with the copper(II) for each dipeptide were obtained over the pH range 4 to 8. In addition, a weighted average of the metal-proton distances between copper(II) and bound solvent molecules in the axial and equitorial positions was obtained. From these measurements and EPR data, a monomeric solution structure for copper(II)-carnosine is proposed. This structure has a β-amino nitrogen, deprotonated peptide nitrogen, α-carboxyl oxygen, and a water molecule in the equitorial plane of copper(II), and an imidazole nitrogen as the fifth ligand in the axial position.

Heme models. I. Solution behavior of a water soluble iron porphyrin.

A well-behaved water soluble iron-porphyrin system, meso-tetra-(4-carboxyphenyl) porphinato iron (III) was synthesized. Its solution behavior is described using visable and electron paramagnetic resonance (EPR) spectroscopy. The complex exists in solution as three distinct forms of bridged dimers, oxo, hydroxo and aquo, with the following pKs: oxo + H+ in equilibrium hydroxo, pK = 9.58; hydroxo + H+ in equilibrium aquo, pK = 6.72. In the presence of excess imidazole the second pK is found to be 7.05. Detailed analysis of the interaction of the hydroxo-bridged form with imidazole is presented. It is found that one dimer unit simultaneously binds two imidazole molecules, with an over-all equilibrium constant log Keq = -1.22. EPR spectra are presented for the various forms of iron-porphyrin discussed.