Robert K. Poole

Nitrate, but not silver, ions induce spectral changes in Escherichia coli cytochrome d

The absorbance maximum (630 nm) of reduced cytochrome d in Escherichia coli membrane particles was diminished by 160 μM AgNO3 or NaNO3 and accompanied by the formation of a species with an absorption maximum at 640–645 nm. Nitrite, trioxodinitrate and nitric oxide elicited qualitatively similar, but faster, changes in the spectrum of cytochrome d, suggesting that formation of a nitrosyl complex may be involved in all cases. In direct contrast to an earlier report, silver ions (160 υM) were without effect on the α‐bands of reduced cytochromes d, b or a 1.

Filamentous growth of Escherichia coli K12 elicited by dimeric, mixed-valence complexes of ruthenium.

Dimeric, mixed-valence [(Ru(II), Ru(III)] compounds of ruthenium caused filament formation in growing cultures of Escherichia coli K12. Three compounds with the general formula Ru2(NH3)6X5 · H2O (where X is a halide) were tested; in order of decreasing effectiveness (and with the concentration giving maximum effect), these were the bromo (10-5M), chloro (10-4 to 10-5M), and iodo (10-3 to 10-4M) analogues. Filamentation elicited by the bromo and chloro compounds was spontaneously reversible after 3–4 h, and tentatively attributed to oxidation of the active mixed-valence form to inactive Ru(III) complexes. Several compounds known to accelerate division of filaments formed under other conditions were ineffective in reversing the filamentation, but the presence of 0,43 M-dimethylsulphoxide totally inhibited filamentation caused by the bromo or chloro compounds and by cis-Pt(NH3)2Cl2 (cisplatin), an established filamenting and antitumour agent. The ruthenium complexes bound to mammalian DNA, but were without effect on the UV spectrum or cellular content of DNA in E. coli, despite showing marked mutagenic activity in reverse mutation tests with Salmonella typhimurium. Cells remained sensitive to inhibition of division by the ruthenium complexes until immediately prior to the division event. Possibilities for the (probably complex) mode of action and the potential of related compounds for therapeutic use are discussed.

Purification and partial characterization of two cytochrome oxidases (caa3 and o) from the thermophilic bacterium PS3.

Two cytochrome oxidases, cytochrome aa3 (EC 1.9.3.1) and cytochrome o, have been purified from the membranes of a thermophilic bacterium, PS3. The enzymes were solubilized with Triton X-100 and purified to apparent homogeneity on anion-exchange columns. The properties of the three-subunit cytochrome oxidase complex caa3 obtained here are compared with the same enzyme isolated by Sone, N. and Yanagita, Y. (1982) (Biochim. Biophys. Acta 682, 216-226). On storage, the purified caa3 enzyme undergoes denaturation; a shoulder at 432 nm seen in (CO-reduced)-minus-reduced difference spectra may be due in part to denaturation products of the enzyme. The purified cytochrome o is more stable. At room temperature, the reduced-minus-oxidized difference spectrum shows absorbance maxima at 427 and 559 nm; at 77 K, its alpha-band is split into 554 and 557 nm components. At room temperature, the CO-reduced-minus-reduced spectrum shows troughs at 430 nm and 560 nm. Dissociating polyacrylamide gel electrophoresis suggests that the purified cytochrome o is composed of one type of subunit with an apparent molecular mass of 47 000-48 000. Metal analysis of the purified enzyme demonstrated the lack of copper. Both oxidases, purified in the presence of Triton X-100, exist in highly polydisperse forms.

Resonance Raman spectroscopy of an oxygenated intermediate species of cytochrome oxidase d from Escherichia coli

Resonance Raman spectroscopy (excitation at 647.1 nm) of solubilized and aerated cytochrome oxidase d from Escherichia coli K12 has shown absorptions (1078–1105 cm−1) attributed to the oxygen—oxygen stretching frequency of the oxidase—oxygen adduct. These findings support the hypothesis that the 650–652 nm chromophore of cytochrome d is an oxygenated or ’oxy’ intermediate species and not the fully oxidized enzyme.

Development of Respiratory Activity during the Cell Cycle of Schizosaccharomyces pombe 972 h-: Respiratory Oscillations and Heat Dissipation in Cultures Synchronized with 2#-Deoxyadenosine

SUMMARY: Rates of oxygen uptake and heat dissipation were measured in cultures of Schizosaccharomyces pombe that had been induced to divide synchronously by adding 2 mM-2#-deoxyadenosine and then removing the inhibitor after 4 h. Respiratory oscillations occurred during the last 1.5h of treatment with deoxyadenosine and throughout the subsequent period of synchronous growth. Before completion of the first synchronous division three peaks of oxygen uptake occurred, the third peak being coincident with cell division. These peaks were less sensitive to the rate-stimulating effect of the uncoupler, carbonyl cyanide m-chlorophenylhydrazone, than were the troughs, so that in the presence of the uncoupler the oscillations were attenuated. In the absence of uncoupler, heat dissipation of the culture increased linearly during and after deoxyadenosine treatment, with sharp increases (approximate doublings) in the rate of dissipation occurring at intervals similar to the mean generation time of an exponential culture. Heat dissipation also increased continuously in samples removed from such a culture and incubated with the uncoupler. The possible modulation of oxygen uptake rates by respiratory control, and the implications of linear increases in heat dissipation are discussed.

Oxidation-reduction potentials and spectral properties of some cytochromes from Thiobacillus versutus (A2)

Cytochromes c-550 (acidic), c-550 (basic), c-551 and c-552.5 from Thiobacillus versutus have been highly purified and characterized. Their spectral properties at 77 K are described. Oxidation-reduction titrations of cytochromes c-550 (acidic) and c-550 (basic) showed them to exhibit Nernst values of n = 1, with single redox centres in the cytochromes, and to have midpoint redox potentials at pH 7.0 (Em,7) of 290 and 260 mV, respectively. Cytochrome c-551 contained two separately titratable redox components, each giving n = 1. The low potential centre (55% of titratable cytochrome) and the high potential centre (45%) had Em,7 values of -115 and +240 mV, respectively. Cytochrome c-552.5 also contained at least two redox centres. One (65% of titratable cytochrome) had n = 1 and Em,7 = 220 mV. The remaining 35% appeared to be a low potential component with an Em,7 possibly as low as -215 mV. the roles of these cytochromes in respiratory thiosulphate oxidation are discussed.

Identification of cytochromes o and a3 as functional terminal oxidases in the thermophilic bacterium, PS3

Low‐temperature photodissociation spectra of membranes from the thermophile PS3 reveal cytochromes o and a 3. The latter reacts with O2 at −103°C to give a light‐insensitive compound(s), but the initial stages of O2 binding to cytochrome o could not be studied under these conditions. Photochemical action spectra identify cytochromes a 3 and o, but not a CO‐binding c‐type cytochrome, as functional terminal oxidases in this bacterium.

LIGAND-BINDING CYTOCHROMES a 3, c and o IN MEMBRANES FROM THE THERMOPHILIC BACTERIUM PS3

The isolation and characterization of a highly stable adenosine triphosphatase from the thermophilic bacterium PS3 [1 ] has given new im.petus to studies of the electron-transferring and energy-transducing proteins from such bacteria. Interest has been heightened by the finding that the cytochrome oxidases of thermophiles and of certain other bacteria bear a remarkable resemblance to the cytochrome c oxidase (EC 1.9.3.1) of eukaryotic mitochondria, but appear to have much simpler subunit compositions [2]. The extreme case appears to be PS3 in which the purified cytochrome oxidase has been reported to have only one subunit, and yet possesses haems a and aa, cytochrome c and intrinsic copper [3]. However, Fee et al. [4] were unable to demonstrate any perturbation of the optical spectra of PS3 membranes by CO, the classical a3-binding ligand. Later experiments (Yang, E. and Sone, N., unpublished; cited in [2]) using low temperature spectroscopy revealed that an a3 component was missing and that the complex contained haems a, o and c in molar ratios 5 : 1 : 5. The purpose of the present paper is to clarify this situation and report the binding of CO, cyanide and NO by cytochromes in membranes isolated from PS3. A ligand-binding a-type cytochrome (thus classifiable as a3 [5]), together with cytochrome o and a ligandbinding cytochrome c are found.

The oxygen reactions of bacterial cytochrome oxidases

Abstract Mitochondrial and bacterial cytochrome oxidases have a common function, the trapping and reduction of oxygen. Cytochrome c oxidase of mitochondria and cytochrome o of Escherichia coli achieve this by kinetically distinct means, each involving the formation of transient and elusive intermediate species. Similar intermediates have been detected in the oxygen reactions of diverse bacterial oxidases.

An apparatus for the continuous and simultaneous monitoring of oxygen and solute uptake by growing microbial cultures: Application to synchronous and asynchronous cultures of Escherichia coli

Abstract A simple apparatus has been constructed for continuous and simultaneous measurements of the uptake of O 2 and radioactively labelled substrates by growing cell cultures. It combines the principles of an O 2 electrode system open to the atmosphere and flow dialysis. Time resolution and sensitivity are appropriate for measurements on small cultures (typically 2–3 ml) of moderate cell density. Measurements on synchronous cultures of Escherichia coli reveal an exponentially increasing, continuous pattern of O 2 uptake, but discontinuities in succinate uptake.