Russell Timkovich

Detection of the stable addition of carbodiimide to proteins

Abstract A simple convenient synthesis is described for radioactive 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. This reagent makes possible convenient detection of the extent of internal rearrangement and the stable addition of carbodiimide to protein when the compound is used in chemical modification studies. A survey of the reaction of carbodiimide alone with eleven typical proteins indicates that the reagent itself may be suitable for selective modification of only a few residues, including carboxylic acid side chains partially buried on the surface or in active sites. This would provide a powerful and selective tool for probing residue function in enzymes.

Isolation of Paracoccus denitrificans cytochrome cd1: Comparative kinetics with other nitrite reductases☆

Abstract The dissimilatory nitrite reductase of the cytochrome cd 1 type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd 1 from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd 1 from another strain using nitrite as ultimate acceptor. Donors included c -type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd 1 , which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd 1 -Alcaligenes c 554 reaction, which was 4-fold faster than the native Alcaligenes cd 1 -Alcaligenes c 554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.

Resonance Raman and surface-enhanced resonance Raman studies of cytochrome cd1.

Cytochrome cd, is the dissimilatory nitrite reductase found in many facultative, anaerobic denitrifying bacteria. It is an oxidation-reduction enzyme that accepts reducing equivalents from donor c-type cytochromes and transfers these to nitrite, reducing it to nitric oxide predominantly [ 11. It reduces oxygen to water [2]. The native enzyme is a dimer composed of two identical subunits each of which is -60 000 Mr and contains one c-type and one d,-type heme [3]. Because of the spectral properties of the prosthetic groups, visible [4-61, electron paramagnetic resonance [7,8] magnetic circular dichroism [9] and emission [lo] spectroscopy have been used to provide structural and physical information about the enzyme. In addition, resonance Raman (RR) spectroscopy is an invaluable technique for providing detailed insights into the structure and environment of heme in hemoproteins. Here we present resonance Raman data for cytochrome cd, and illustrate the scope of this approach. ties among these proteins. SERRS has been used here to obtain preliminary heme structure/environment comparisons between Pseudomonas and Paracoccus cytochromes cd,. Because of the following points of similarity, cytochrome cdl can be viewed as an analogue for mitochondrial cytochrome aa3: cdl is a cytochrome c oxidase; it can reduce O2 in vitro; heme d, is spectroscopically related to heme a. Spectra reported here for cd, will be compared to data available for c and aa to test the extent of analogy.

Evidence for water as the product for oxygen reduction by cytochrome cd

Abstract Evidence is presented that water is the final product of electron donation to molecular oxygen by cytochrome cd from Paracoccus denitrificans when ferrocytochrome c acts as donor to cd . Negative evidence for the accumulation of superoxide and peroxide was obtained by rate effect experiments in the presence of superoxide dismutase, catalase, and peroxidase. Positive evidence for water was obtained by showing a 4 to 1 stoichiometric balance for rates of electron acceptance from ferrocytochrome c to rates of donation to molecular oxygen.

Purification and properties of Paracoccus denitrificans Azurin.

Abstract The isolation of an azurin type Cu protein from Paracoccus denitrificans (ATCC 13543) is described and some properties are reported. The purified protein has a molecular weight of 13,790 in a single polypeptide chain and contains one Cu atom per molecule. Its spectrum is typical of Type I, “blue” Cu proteins in showing an intense band at 595 nm; but it also shows a weaker absorption band at 448 nm. Its standard reduction potential has been measured to be +230 mV, which is the lowest potential observed to date for azurins isolated from bacterial sources. The purified protein shows fivefold greater electron transport activity with membrane fragments than with the soluble nitrite reductase of Paracoccus . This argues against the latter as the primary physiological oxidase system for azurin.

Polymerization side reactions during protein modifications with carbodiimide.

Abstract A survey has been made of carbodiimide reactions with four typical proteins to reveal that covalent polymerization can be a significant artifact in carbodiimide related chemical modification studies on proteins.

Reaction of redox proteins at a 4,4′-bipyridyl-modified carbon paste electrode

Abstract An oil carbon paste 4,4′-bipyridyl chemically modified electrode has been observed to enhance the electrochemical response in cyclic voltammetry of Pseudomonas azurin, Pseudomonas cytochrome cd1, horse heart cytochrome c, and ferricyanide. Both azurin and ferricyanide adsorb on the surface of the modified electrode. A qualitative model of this electrode is discussed that assumes the existence of a 4,4′-bipyridyl-oil layer on the carbon surface into which substances may diffuse from the solution.

Cyanide reactivity of cytochrome c derivatives.

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivative demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10-20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 . 10(-2) s-1.

Patterns of product inhibition for bacterial nitrite reductase.

Product inhibition has been examined in the turnover kinetics of cytochrome cd1 from Pseudomonas aeruginosa (ATCC 19429) and from Paracoccus denitrificans1 (ATCC 13456). A common characteristic was a decrease in rate during the time course of assays that was not due to substrate depletion or irreversible inactivation. The product of nitrite reduction, nitric oxide (NO), acted as a product inhibitor in anaerobic assays with an apparent Ki of 0.2 microM, but only if the enzyme was first preincubated with NO for 15 min. The enzyme was inhibited by the oxidized form of electron donors and this could account for the decrease in rate during an assay. For the donors hydroquinone, ascorbate, TMPD, and azurin, measured values of the inhibition constant were at least ten fold lower than measured Kms. Cytochromes c as donors demonstrated a complex pattern of product inhibition by the ferric form. Although numerical values of Ki in these cases were not obtained, trends indicated that apparent values would be less than Km.

Magnetic sysceptibility measurements on Pseudomonas cytochrome cd1

The magnetic susceptibilities of cytochrome cd1 from Pseudomonas aeruginosa (American Type Culture Collection 19429) have been measured by a nuclear magnetic resonance technique. In the oxidized form both heme c and heme d1 are in the low-spin state with an average magnetic moment of 2.6 Bohr magnetons. At 25 degrees C and pH 8.0, the ascorbate-reduced cytochrome contains one low-spin and one high-spin heme per subunit. Based on previous reports in the literature, the high-spin ferrous heme has been assigned to the heme d1 group. At pH 8.0 the ascorbate-reduced heme d1 has a magnetic moment of 5.3 Bohr magnetons. This value decreases to 4.9 at pH 5.5, but is still indicative of a high-spin ferrous system. The paramagnetic susceptibility of the ferricytochrome demonstrated a temperature dependence consistent with Curies law, but the ferrocytochrome showed an increase in paramagnetic susceptibility with increasing temperature.