Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D -galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA (cellulase synthase-like protein). In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective, the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.

The steady-state behaviour of isolated mammalian cytochrome c oxidase was examined by increasing the rate of reduction of cytochrome c . Under these conditions the enzyme's 605 (haem a ), 655 (haem a 3 /Cu B ) and 830 (Cu A ) nm spectral features behaved as if they were at near equilibrium with cytochrome c (550 nm). This has implications for non-invasive tissue measurements using visible (550, 605 and 655 nm) and near-IR (830 nm) light. The oxidized species represented by the 655 nm band is bleached by the presence of oxygen intermediates P and F (where P is characterized by an absorbance spectrum at 607 nm relative to the oxidized enzyme and F is characterized by an absorbance spectrum at 580 nm relative to the oxidized enzyme) or by reduction of haem a 3 or Cu B . However, at these ambient oxygen levels (far above the enzyme K m ), the populations of reduced haem a 3 and the oxygen intermediates were very low (<10%). We therefore interpret 655 nm changes as reduction of the otherwise spectrally invisible Cu B centre. We present a model where small anti-cooperative redox interactions occur between haem a –Cu A –Cu B (steady-state potential ranges: Cu A , 212–258 mV; haem a , 254–281 mV; Cu B , 227–272 mV). Contrary to static equilibrium measurements, in the catalytic steady state there are no high potential redox centres (>300 mV). We find that the overall reaction is correctly described by the classical model in which the Michaelis intermediate is a ferrocytochrome c –enzyme complex. However, the oxidation of ferrocytochrome c in this complex is not the sole rate-determining step. Turnover is instead dependent upon electron transfer from haem a to haem a 3 , but the haem a potential closely matches cytochrome c at all times.

Ferryl (Fe(IV)=O) species are involved in key enzymatic processes with direct biomedical relevance; among others, the uncontrolled reactivities of ferryl Mb (myoglobin) and Hb (haemoglobin) have been reported to be central to the pathology of rhabdomyolysis and subarachnoid haemorrhage. Rapid-scan stopped-flow methods have been used to monitor the spectra of the ferryl species in Mb and Hb as a function of pH. The ferryl forms of both proteins display an optical transition with p K ∼4.7, and this is assigned to protonation of the ferryl species itself. We also demonstrate for the first time a direct correlation between Hb/Mb ferryl reactivity and ferryl protonation status, simultaneously informing on chemical mechanism and toxicity and with broader biochemical implications.

The paramagnetic species in human metHb and horse metmyoglobin (metMb) have been studied at low temperature using EPR spectroscopy. The high-spin (HS) haem signal in aquometMb has a greater rhombic distortion than the HS metHb signal. Nevertheless, the individual line width ( g = 6) is smaller in metMb than in metHb, consistent with non-identical signals from the α and β Hb subunits. Three low-spin (LS) haem forms are present in metHb, while metMb has only two. The major LS form in both proteins is the alkaline species (with OH - at the sixth co-ordination position). The minor LS forms are assigned to different histidine hemichromes in equilibrium with the normal HS species at low temperature. LS forms disappear when the haem is bound by a ligand, such as fluoride, which ensures 100% occupancy of the HS state both at room temperature and at 25K. The small differences in effective g -factors of the histidine hemichromes are interpreted in terms of different distances between the distal histidine and haem iron. The pH dependence of the inter-conversion of the different paramagnetic species is consistent with a model whereby protonation of a residue with a p K of 5.69 (metHb) or 6.12 (metMb), affects ligand binding and transformation from the HS to the LS form. Chemical and spectroscopic considerations suggest that the residue is unlikely to be the proximal or distal histidine. We therefore propose a model where protonation of this distant amino acid causes a conformational change at the iron site. Identical effects are seen in frozen human blood, suggesting that this effect may have physiological significance.

The control of cytochrome c oxidase turnover in proteoliposomes by membrane potential (ΔΨ) and by pH gradient (ΔpH) is probably kinetic in nature, and inhibition by valinomycin and stimulation by nigericin indicate that ΔpH exerts a greater influence than does an equivalent ΔΨ. Oleic acid at 100 µ M removes all ΔΨ and ΔpH control, whereas a similar concentration of palmitic acid increases turnover but does not completely abolish control. Valinomycin acts synergistically with both fatty acids, indicating that the latter can act as H + /K + exchangers, but neither fatty acid alone markedly affects ΔpH, showing that they cannot fully mimic nigericin. Oleate, but not palmitate, diminishes ΔΨ, and can move electrophoretically as oleate anion. Submicromolar palmitic acid concentrations partly stimulate turnover in ΔΨ- and ΔpH-controlled proteoliposomes, as reported by Labonia, Muller and Azzi [(1988) Biochem. J. 254 , 130–145], which might represent a direct effect on cytochrome c oxidase. The ubiquity of fatty acids in biological membranes suggests that these substances might be responsible for limiting respiratory control and enzyme activity in vivo .

Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p -trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1′-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN′N′ -Tetramethyl- p -phenylenediamine does not behave as a simple electron carrier. During NNN′N′ -tetramethyl- p -phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN′N′ -tetramethyl- p -phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN′N′ -tetramethyl- p -phenylenediamine is used as a mediator.

1. On addition of reductant (ascorbate plus NNN′N′ -tetramethyl- p -phenylenediamine) to isolated cytochrome c oxidase (ox heart cytochrome aa 3 ), in the presence of the inhibitors azide or cyanide, an initial partially reduced species is formed with absorption peaks at 415nm, 445nm and 605nm, which slowly gives rise to the final ‘half-reduced’ species in whose spectrum the 415nm peak has disappeared and a new absorption is seen at 430–435nm. 2. In the absence of reductant, cyanide forms an initial complex with the enzyme with a spectrum similar to that of the uncombined form, which slowly changes into the ‘low-spin’ cyanide form with a peak at 432nm. Azide, in absence of reductant, shifts the Soret peak slightly, but the resulting complex, which is probably thermally ‘mixed-spin’, undergoes no further changes. 3. The Soret-peak shift of oxidized cytochrome a 3 which occurs on reduction of the enzyme in the presence of azide is accompanied by a concurrent blue shift of the ferrous cytochrome a peak from 605nm to 603nm. A partial blue shift of the α-peak occurs in the half-reduced sulphide-inhibited enzyme, and a complete blue shift is seen in the analogous complexes with alkyl sulphides [ a 2+ a 3 3+ HSR compounds, where R=CH 3 , C 2 H 5 or (CH 3 ) 2 CH]. 4. Analogous, albeit less readily decipherable, spectroscopic effects with the ligands imidazole and alkyl isocyanides suggest that on reduction of cytochrome a an interaction occurs between the two haem groups involving (i) a high- to low-spin change in cytochrome a 3 , and after this, (ii) a change in the molecular environment of the cytochrome a . The latter effect, possibly a decrease in the hydrophobicity of the haem pocket, requires that the ligands on cytochrome a 3 have a bulky and partially hydrophobic character.

Lineweaver–Burk plots of 1/ v against 1/[O 2 ] for rat liver mitochondrial respiration with succinate or ascorbate+ NNN′N′ -tetramethyl- p -phenylenediamine as substrates are non-linear. In state 3u (uncoupled by trifluoromethoxycarbonyl cyanide phenylhydrazone) such plots tend to be concave upward, whereas in state 4 (energized) the plots were concave downward. The apparent K m for oxygen is larger in state 4 than in state 3u, despite the higher turnover in the latter system. It is postulated that at least one reversible reaction occurs between cytochrome c and cytochrome c oxidase, whose rate is increased on energization (reversed electron transfer); a model including such a reaction is proposed which accounts semiquantitatively for the observations.

1. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are described. Kinetic differences between the older and more recent preparations of the enzyme most probably arise from differences in intrinsic turnover rates. 2. The time-courses of cytochrome c peroxidation by the enzyme follow essentially first-order kinetics in phosphate buffer. Deviations from first-order kinetics occur in acetate buffer, and are due to a higher enzymic turnover rate in this medium accompanied by a greater tendency to autocatalytic peroxidation of cytochrome c . 3. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are interpreted in terms of a mechanism postulating formation of reversible complexes between the peroxidase and both reduced and oxidized cytochrome c . Formation of these complexes is inhibited at high ionic strengths and by polycations. 4. Oxidized cytochrome c can act as a competitive inhibitor of ferrocytochrome c peroxidation by peroxidase. The K i for ferricytochrome c is approximately equal to the K m for ferrocytochrome c and thus probably accounts for the observed apparent first-order kinetics even at saturating concentrations of ferrocytochrome c . 5. The results are discussed in terms of a possible analogy between the oxidations of cytochrome c catalysed by yeast peroxidase and by mammalian cytochrome oxidase.