Haywood Blum

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.

A transmembrane quinone pair in the succinate dehydrogenase-cytochrome b region

Recently, Ruzicka et al. [l] reported an EPR signal characteristic of dipolar coupled species in beef heart mitochondria. At first this signal [2] was believed to be due to the interaction of ubisemiquinone with an iron sulfur center, but simulation of the spectra in Complex II [l] showed that a better fit could be obtained if the other species &as another semiquinone. Potentiometric methods confirmed this and the second species was identified as a ubisemiquinone rather than a flavosemiquinone [3,4] which had been considered a viable alternative [l] . The orientation of the heme planes in cytochrome oxidase has recently been determined using oriented multilayers made by centrifugation and partial drying of purified cytochrome oxidase vesicles, electron transport particles [5] and mitochondria [6] . The necessary assumption is that each molecule (and therefore each heme) has a relatively futed orientation in the direction normal to the membrane; this is borne out by experimental results. Analysis of the EPR spectra of the coupled quinone pair in oriented multilayers made from mitochondria allow information about the orientation and relative location of the quinones in the membrane to be deduced.

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.

The orientation of a heme of cytochrome c oxidase in submitochondrial particles

The electron paramagnetic resonance of the low spin signal from oxidized cytochrome c oxidase has been studied in oreinted multilayers of submitochondrial and electron transport particles. Measurements of the angular variation of the EPR spectra with the multilayer plane orientation allow the determination of the heme orientation in the multilayer. The heme normal lies in the membrane plane and the y-axis of the heme makes an angle of 30 degrees with the membrane normal. Analysis of the line shape reveals the presence of mosaic spread in the multilayer almost half of which is attributable to deviations of protein orientation within the membrane.

Electron spin relaxation of the electron paramagnetic resonance spectra of cytochrome c

The progressive power saturation of the electron paramagnetic resonance (EPR) spectrum of ferricytochrome c has been investigated in order to determine the spin-lattice relaxation time of the center. We have generalized the usual saturation treatments to include the effects of extended sample size and anisotropic g values as well as derivative spectra. We find that the results are consistent with a T7 power law in the temperature range 6--25 K. At temperatures above 25 K the relaxation time is too short for successful power saturation. Observation of the linewidth shows that the relaxation behavior continues as a first-order Raman process to 50 K.

The orientation of bovine adrenal cortex cytochfome P-450 in submitochondrial particle multilayers

Angular electron paramagnetic resonance spectra of cytochrome P-450 in oriented multilayers from bovine adrenal cortex Submitochondrial particles have been obtained. Both high- and low-spin forms are present. Analysis of the spectra allows the orientation of the cytochrome P-450 to be determined relative to the membrane plane. The quality of the orientation of the cytochrome P-450 is estimated by use of a computer simulation program. Cytochrome P-450, unlike other cytochromes and porphyrins previously studied, has its heme plane parallel to the membrane plane.

Determination of the exchange integral in binuclear iron-sulfur clusters in proteins of varying complexity.

The coupling constants J between the iron atoms in ferredoxin type iron-sulfur proteins containing binuclear clusters were evaluated by two parallel methods. The temperature dependence of the EPR linewidths and integrated abosrption intensities are both related to the energy of the first excited state. The values of J obtained were: center S-1 in succinate dehydrogenase, 90 cm-1; Rieskes iron-sulfur center, 65 cm-1; adrenodoxin, 270 cm-1. The behavior of iron-sulfur center N-1a in NADH:UQ reductase was also examined; its similarity to that of center S-1 indicates that center N-1a is also a binuclear iron-sulfur center, with J = 90 cm-1. Greater rhombic distortion present in the EPR spectrum of a binuclear cluster was associated with smaller values of J.

A model for the simulation of the EPR spectra of chromophores in partially oriented membrane multilayers

Abstract The EPR spectrum of membrane-bound chromophores in oriented membrane multilayers can be simulated by a model which assumes a rhombic g tensor, an angle-dependent linewidth, and a fixed orientation of the center relative to the membrane normal. Membrane multilayers display no order with respect to rotation about the membrane normal. The remaining order- is subject to mosaic spread. Application of appreciable mosaic spread causes the line positions to move from their expected values to the principal g values and the lineshapes change from derivative to absorption-like powder spectra.

Effect of dysprosium on the spin-lattice relaxation time of cytochrome c and cytochrome a.

The progressive power saturation of the electron paramagnetic resonance of horse heart cytochrome c and solubilized bovine heart cytochrome oxidase has been monitored at low temperature in the presence of the relaxing agent, dysprosium. The saturation of the EPR signal of cytochrome c is relieved even at 6 K. With increasing temperature the effect is enhanced as the relaxation time of the dysprosium becomes shorter; however, the intrinsic spin-lattice relaxation time, T1, for cytochrome c decreases even more rapidly with increasing temperature. T1 for cytochrome c can be described by an intrinsic component, a component which is proportional to the concentration of dysprosium and a third component due to local binding which is independent of dysprosium concentration. The cytochrome a component of cytochrome oxidase is also affected by dysprosium. In the presence of cytochrome oxidase, T1 for cytochrome c is almost unaffected by dysprosium, indicating that access to the cytochrome c heme is blocked by the binding of c to oxidase. Based on the concentration-dependent effect of dysprosium on the lifetime of cytochrome c, it is possible to make distance estimates from the EPR active center to Dy3+. Dysprosium is therefore useful for determining the spatial relationships among paramagnetic enzyme components in a quantitative way.

Applications of Dextran-magnetite as a sodium relaxation enhancer in biological systems

Dextran-magnetite particles consist of macromolecular magnetite (Fe304) cores coated with the hydrophilic polymer dextran. These particles are completely soluble in aqueous solution and are similar to the iron-dextran complex which has been used to stimulate hematopoesis (I). However, unlike iron-dextran particles, dextran-magnetite particles are superparamaguetic and possess a large, inducible magnetic moment. In magnetic fields over a few thousand gauss, this magnetic moment approaches the saturation magnetization of 5660 G for ferromagnetic magnetite (2). This magnetic moment is two to three orders of magnitude larger than that for similarly sized paramagnetic materials at commonly used NMR field strengths (3). Almost a decade ago, dextran-magnetite was shown to be at least ten times more effective on a molar basis than the Mn2’ ion in reducing the T2 of water protons (4). Since this initial report, there has been relatively little use of this reagent in spectroscopic studies. Part of the reason for this may have been the fact that the reagent was not readily available. The particles used in the initial report were specially prepared and have not been commercially marketed. Recently, however, a simple synthetic procedure has been developed and published (5). Essentially, this protocol involves the precipitation of dextran-magnetite from a stoichiometric mixture of ferrous and ferric chlorides in a viscous dextran solution. The completely soluble particles possess a dense magnetite core ranging from 100-200 A in diameter and are approximately 30% dextran by weight. In addition to relaxing water protons, dextran-magnetite also effectively decreases the T2 of sodium in aqueous solution. In Fig. IA, the 23Na spectrum from a 140 nUI4 NaCl solution containing dextran-magnetite at an approximate molar particle concentration of 3.0 ti (14.5 g magnetite/liter) is displayed. This spectrum was recorded using a Bruker CXP-200 spectrometer operating at 52.9 MHz for sodium; the observed linewidth is slightly greater than 6900 Hz. In contrast, a well shimmed saline sample has a linewidth of only 20 Hz under the same conditions. Figure 1B also contains a spectrum obtained from fresh human erythrocytes which have been washed in 140 mM NaCl containing 3.0 N dextran-magnetite particles. Due to the relatively large size of the particles, they do not enter cells in solution. The resulting spectrum is readily seen to contain two components; a larger, broader component arising from the extracellular sodium and a smaller, narrower component arising from the intra-