H.A.O. Hill

Enhanced production of hydroxyl radicals by the xanthine-xanthine oxidase reaction in the presence of lactoferrin

The generation of hydroxyl radicals by the xanthine-xanthine oxidase reaction (C. Beauchamp and I. Fridovich (1970) J. Biol. Chem. 245, 4641-1616) has been shown to be increased by iron-saturated lactoferrin isolated from pig neutrophils. Hydroxyl radical production, measured by EPR spin trapping and by ethylene production from alpha-keto-gamma-methiol butyric acid, has been demonstrated to be produced by a Fenton-type Haber-Weiss reaction catalysed by lactoferrin. The possibility that lactoferrin catalyses such a reaction in vivo is considered.

The effect of pH and temperature on the structure of the active site of azurin from Pseudomonas aeruginosa

1. INTRODUCTION The unusual spectroscopic properties of the blue copper proteins, such as azurin and plastocyanin, have attracted much attention [ 1,2]. The success of spectroscopic methods in predicting the coordina- tion environment of the copper could be assessed on publication of the crystal structures of a plastocya- nin [3] and an azurin [4,5]. It appears that in poplar plastocyanin, the Cu-atom is coordinated by two histidines (residues 37 and 87), a cysteine (Cys 84) and a methionine (Met 92) in a distorted tetrahedral configuration 131. Although the resolution of the crystal structure of azurin is somewhat less, the data suggest that the Cu is coordinated by two histidines (His 46 and 117) a cysteine (Cys 112) .and a methionine (Met 121), again in a distorted tetra- hedral configuration. Possible contributions from other ligands, e.g., from the peptide backbone, could not be completely excluded [5]. of magnitude slower in exchanging electrons cyto- chrome c-555 than the former. The redox potential of the ‘inactive’ form is 60 mV lower than that of the ‘active’ form, indicating a stabilization of the Cu(I1) state towards high pH [6-81. The transition from active to inactive state involves deprotonation of the histidine [6] which was shown in NMR studies [9- 121 to participate in a slow proton-exchange process. In a recent NMR study [ 131 this residue has been as- signed to His 35, which is adjacent to the ligand His 46 [4,5]. We wish to report the results of some NMR ex- periments on

Electrochemical reduction of dioxygen using a terminal oxidase

The reduction of dioxygen to water is a reaction central to aerobic life. Much is known about the terminal oxidases of both prokaryotes and eukaryotes. They contain at least two redox centres and are efficiently coupled to the redox proteins which preceed them in the electron-transport chain. Coincidentally with the interest in the biological reduction of dioxygen to water, much effort has been expended [l] in the search for efficient ‘oxygen’ electrodes. Nonenzymic reduction of dioxygen to water is ]2] often slow and usually involves a complex series of steps. Elsewhere we have [3,4] drawn attention to the possible parallels between electron transport at an electrode surface and that in biological systems. We have reported [5] an attempt to couple electron transport between a surface-nlodi~ed gold electrode and a nl~rn~ian cytochrome oxidase. We now describe the successful coupling of electron transport between a gold electrode, upon which 1,2-bis(4-pyridyl)ethene is adsorbed j&-9], and the soluble terminal oxidasef nitrite reductase of Psez~dorno~as ae~~g~nosa.

N-(2-Ferrocene-ethyl)maleimide : a new electroactive sulphydryl-specific reagent for cysteine-containing peptides and proteins

We report the synthesis and application of a specific electroactive label, N‐(2‐ferrocene‐ethyl)maleimide, which provides new redox properties to organic compounds and proteins possessing sulphydryl groups. Its reaction conditions with the cysteine‐containing peptide, glutathione, and a terminal monooxygenase enzyme, cytochrome P450cam are presented. The labelled peptide and enzyme acquired reversible electrochemical properties due to the attached ferrocene moiety.

Does caeruloplasmin dismute superoxide? No

Caeruloplasmin (ferroxidase, EC 1 .16.3 .l) is a copper containingplasmaa&ycoprotein.Three biological functions have been ascribed to caeruloplasmiri: (a) ferroxidase activity; (b) copper transport and Storage; and (c) maintenance of copper homeostasis in;the tissues. In [l] another function for caeruloplasmin has been proposed. These workers have demohstrated that caeruloplasmin inhibits the reduction of ferricytochrome c and of nitroblue tetrazolium by superoxide produced by the aerobic action of xanthine oxidase on hypoxanthine. Consequently it was proposed that caeruloplasmin may perform the function of scavenging any superoxide that leaks in the plasma where the levels of superoxide are extremely low. Here we report that caeruloplasmin does not catalyse the disproportionation of superoxide anion radicals generated by pulse radiolysis. However, the reduction of the type 1 copper(I1) in caeruloplasmin is observed.

Site‐specific introduction of an electroactive label into a non‐electroactive enzyme (β‐lactamase I)

A cysteine residue was introduced close to the active site of β‐lactamase I by site‐directed mutagenesis to replace tyrosine‐105 and was subsequently modified with an electroactive SH‐specific reagent, N‐(2‐ferrocene‐ethyl)maleimide. The resulting modified enzyme became electroactive, showing good quasi‐reversible electrochemistry which was characteristic of the attached ferrocene moiety while retaining its specific enzymatic activity. In the presence of a suicide substrate, 6β‐iodopenicillanic acid, the redox potential shifted +20 mV suggesting that the label was sensitive to changes in the active site of the enzyme.

A proton NMR study of the electron exchange between reduced and oxidized azurin from Pseudomonas aeruginosa

Abstract The line broadening observed at 50C in the proton NMR spectrum of a partly oxidized solution of azurin from Pseudomonas aeruginosa is consistent with assignments of peaks belonging to the ligand residues His-46, His-117, and Met-121 of the copper atom. From the analysis of the tine broadening a value of k = 2 × 106 M− sec−1 is obtained for the bimolecular rate constant of the electron self-exchange reaction of azurin at 50°C. The analysis also provides insight into the spin density distribution over the Cu ligands in oxidized azurin. Finally the data show that small configurational changes in the vicinity of Val-31 occur upon oxidation of the protein.

Direct electrochemical oxidation of Clostridium pasteurianum ferredoxin: Identification of facile electron-transfer processes relevant to cluster degradation

Rapid oxidation processes relevant to the degradation of [4Fe‐4S] clusters in Clostridium pasteurianum ferredoxin were studied via direct (unmediated) heterogeneous electron transfer at a pyrolytic graphite electrode. Differential‐pulse voltammograms of native [4Fe‐4S] ferredoxin showed two well‐defined oxidation peaks corresponding to apparent E‐values of +793 and +1120 mV at 5°C. Direct involvement of the cluster was established through parallel experiments with the 2[4Fe‐4Se] derivative for which peak positions were shifted. Square‐wave voltammetry showed that the product of the first electron transfer, which may correspond to the ‘super‐oxidised’ [4Fe‐4S]3+ oxidation level, undergoes rapid degradation (t ½ < 1.6 ms at 5°C). The second oxidation process, as characterised by a significant (⪢100 mV) negative shift upon selenium substitution, very likely represents oxidation of S(Se) still associated with the protein and possibly contained within the remaining FE‐S(Se) substructure.

The inhibition of the vitamin K-dependent carboxylation of glutamyl residues in prothrombin by some copper complexes.

The bios~thesis of prothrombi~ and the other plasma clotting factors VII, IX and X, involves a vitamin K-dependent post-translational modification in which specific glutamyl residues are carboxylated on the y-carbon to give r-carboxyglutamyl residues [ 1,2]. The reaction requires the vitamin in its reduced form (vitamin K hydroqu~none), molecular oxygen and carbon dioxide ]3,4] together with a carboxylase system from liver microsomal fractions. Vitamin Kr quinone is also active if added with NADH, the reduction being accomplished by an NADH-dependent vitamin K, reductase present in the liver microsomes [.5]. The substrate for the reaction is either the hepatic prothrombin precursor that accumulates during vitamin Kr deficiency in rats [6,7] or a pentapeptide based on residues 5-9 of prothrombin [S]. The reaction does not require ATP ]4,9] or biotin ilO]It has been shown [I I] that vitamin Kr semiquinone will reduce dioxygen to superoxide and that liver microsomes produce [ 121 superoxide by an NADPH-dependent process. We have shown [ 131 that the N~H~ependent generation of superoxide from normal rat liver microsomes is stimulated by the addition of vitamin Kr and that superoxide dismutase (SOD) (10 pg/ml) scavenges the superoxide which is produced. We have also shown that carboxylation of the prothrombin precursor and pentapeptide is depressed by SOD, but only at 31 mg SODjml, This inefficiency of the superoxide dismutase in preventing carboxylation may be explained by the inability of the enzyme to reach the superoxide generating site. It

The redox properties of azurin from pseudomonas aeruginosa as studied by high frequency proton NMR

Since their 3-dimensional structures have become available [1–3] the blue copper proteins plastocyanin (Pc) and azurin (Az) have gained increased attention from spectroscopists [4]. In both proteins the Cu atom appears to be coordinated by 2 histidines, a cysteine an a methionine, and also in other respects the two structures exhibit a remarkable similarity. Yet in their electron transfer properties and their conformational behaviour as a function of pH Az and Pc show quite notable differences. In Pc the Cu-coordinating His-87 becomes protonated at low pH (pH < 5.4) [1] and moves away from the metal, thus producing a 3-coordinated copper(I) centre. This leads to stabilization of the Cu(I)- state and loss of redox-activity. Moreover, Pc exhibits a large variation in the rate of electron transfer with other proteins (including itself) [5–7], which has been considered as indicative of specific protein-protein interactions. The existence of charged patches on the protein surface is in accordance with the purported electrostatic nature of these interactions [5]. In the case of Az, on the other hand, it was only known, that the protein undergoes a switch from a redox inactive to a redox active state when the pH drops below about pH = 7 [8]; we therefore started a proton NMR study of the redox properties of Az, the results of which are summarized here. By studying the effect of slight oxidation on the NMR spectrum of Az from Pseudomonas aeruginosa the proton signals of the ligand residues His-46, His-117 and Met-121 were identified and subsequently their pH behaviour was studied [9]. As an example the case of Met-121 is shown here. The signal (labelled M6) of the ϵ-methyl group of this residue at low pH appears at −0.05 ppm from TSS. The unusual upfield shifted resonance position is the result of the combined ring current effects of Phe-15 and His-46. With increasing pH the signal slowly loses intensity and at pH > 8 has disappeared. This was ascribed tentatively to a weakening of the CuS (Met-121) bond and increased motional freedom of the Met-121 methyl group leading to a broadening of the NMR-signal [9]. Our recent experiments show that when peak M6 disappears a new singlet appears (labeled M′6) at a 0.11 ppm that overlaps with the Cγ1-methyl triplet signal of Ile-7 (labeled R5). The single nature of M′6 is demonstrated in the 2-dimensional J-resolved spectrum reproduced in the figure. It is clear therefor, that a conformational change of the protein occurs when the pH is varied, which affects the position of theϵ-methyl group of Met-121 with respect of Phe-12 and/or His-46. Saturation transfer experiments, to be detailed elsewhere, prove that the signals M6 and M′6 are in slow exchange. Further study of the other ligand signals provides additional evidence that Az may exist in two conformations which interconvert on a time scale of 10–100 ms and that the residues His-35, His-46 and Met-121 are involved in the interconversion. On combining the crystallographic and the NMR data, it transpires that the function of His-35 probably is that of a pH-dependent relay between the Cu coordination shell and the protein. In the course of the oxidation experiments it became apparent that the broadening of the NMR signals induced by the paramagnetism of the Cu(II) species would yield information about the rate of electron self exchange of Az. A thorough analysis of the experimental data showed that, for a few NMR signals, the broadening could be analyzed according to the ‘strong pulse limit’. In this limit the broadening is completely determined by the lifetime of the protein in the diamagnetic state and this leads directly to a value of the rate of electron self exchange, k. Although not very accurate (estimated accuracy ±50%) the value of 2 × 106M−1 s−1 found at 50 °C for k in this way [10] clearly is in agreement with the self exchange rate inferred by Wherland and Pecht from the rate of electron transfer between Az and a variety of other redox proteins [6], though not with the data calculated by Gray an coworkers on the basis of a Marcus treatment of the heterogeneous electron transfer between Az and a series of inorganic transition metal compounds [11]. The high rate of self exchange of Az as well as the relatively fast electron exchange between Az and other redox proteins seems to indicates that nonspecific hydrophobic interactions govern the reaction of Az with its reaction partners. This is consistent with the findings from Cr titration experiments [12] and with conclusions from the crystallographic work [2], that there are no pronounced charged patches on the Az surface.