The aerobic respiratory chain of Escherichia coli has two terminal quinol oxidases: cytochrome bo and cytochrome bd. Cytochrome bd was thought to function solely to facilitate micro-aerobic respiration. However, it has recently been shown to be overexpressed under conditions of nitric oxide (NO) stress; we show here that cytochrome bd is crucial for protecting E. coli cells from NO-induced growth inhibition by virtue of its fast NO dissociation rate.

NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and͞or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a 3͞CuB). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been suggested that two NO molecules can interact with the enzyme simultaneously. We used steady-state and kinetic modeling techniques to reevaluate NO inhibition of CCO. At high flux and low oxygen tensions NO interacts predominantly with the fully reduced (ferrous͞cuprous) center in competition with oxygen. However, as the oxygen tension is raised (or the consumption rate is decreased) the reaction with the oxidized enzyme becomes increasingly important. There is no requirement for NO to bind to the singly reduced binuclear center. NO interacts with either ferrous heme iron or oxidized copper, but not both simultaneously. The affinity (K D) of NO for the oxygen-binding ferrous heme site is 0.2 nM. The noncompetitive interaction with oxidized copper results in oxidation of NO to nitrite and behaves kinetically as if it had an apparent affinity of 28 nM; at low levels of NO, significant binding to copper can occur without appreciable enzyme inhibition. The combination of competitive (heme) and noncompetitive (copper) modes of binding enables NO to interact with mitochondria across the full in vivo dynamic range of oxygen tension and consumption rates.bioenergetics ͉ mitochondria ͉ signaling N O is a physiological signaling molecule produced in vivo by enzymes of the NO synthase family. The NO signaling pathway is classically mediated by activation of soluble guanylate cyclase (1-3). However, in 1994 it was additionally shown that mitochondrial oxygen consumption by cytochrome c oxidase (CCO) is reversibly inhibited by NO in a manner apparently competitive with the oxygen tension (4-6). Inhibition of mitochondrial respiration by NO at CCO has since been implicated in a wide range of physiological processes (7-12), and several of these require a competitive (with respect to oxygen) element to the interaction, e.g., activating hypoxia-inducible factor (13); maintaining flow-metabolism coupling in the brain (14); and allowing cells distant from capillaries to maintain adequate oxygenation (15). Under pathological conditions such as sepsis, the elevated NO concentration, generated by inducible NO synthase, is associated with mitochondrial dysfunction and is implicated in mortality in the critically ill (16).However, the detailed molecular mechanism of CCO inhibition by NO has not yet been unequivocally established. The oxygen-binding center of CCO is bimetallic, and dioxygen reacts at this site only when both the heme a 3 iron and the adjacent copper (Cu B ) are reduced (17). Functional studies initially suggested that NO inhibits by...

We re-determined the near infrared (NIR) spectral signatures (650–980 nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980 nm is cupric CuA, which in the beef heart enzyme has a maximum at 835 nm. The 655 nm band characterises the fully oxidised haem a3/CuB binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting ‘perturbed’ binuclear centre is also characterised by a previously unreported broad 715–920 nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835 nm maximum or the 715 nm band. Our results confirm the ability of NIRS to monitor the CuA centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a3/CuB binuclear centre.

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to noniron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.Biodegradation of lignocellulose by microorganisms plays an important role in carbon cycling, is of biotechnological interest to the paper industry, and has potential application in the field of bioremediation. Some microorganisms secrete a range of enzymes that completely degrade all the components of lignocellulose (lignin, hemicelluloses, and cellulose), while others secrete a narrower range of enzymes that only partially achieve this degradation (8,19,21). White rot fungi secrete both cellulolytic and ligninolytic enzymes; heme enzymes are major components of this ligninolytic activity and include the wellcharacterized lignin and manganese peroxidases of Phanerochaete chrysosporium (12).Some actinomycetes, including thermophilic species and streptomycetes, secrete cellulose-and hemicellulose-degrading enzymes (2, 4, 11). Since the discovery of extracellular lignindegrading heme peroxidases of wood-rotting fungi, considerable effort has been expended in searching for analogous enzymes in the cellulolytic actinomycetes (9,22,24). Indeed, some proteins secreted by the actinomycetes Streptomyces thermoviolaceus (17), Streptomyces viridosporus T7A (6, 26, 27), and Thermomonospora fusca BD25 (recently reclassified as Thermobifida fusca [35]) (3, 28) have low peroxidase activity and have been isolated and partially characterized. The peroxidase-like proteins from S. thermoviolaceus and S. viridosporus T7A were assigned as heme peroxidases on the basis of their optical spectra. H...

Graphical abstract

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 a3/CuB) and 830 (CuA) 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 a3 or CuB. However, at these ambient oxygen levels (far above the enzyme Km), the populations of reduced haem a3 and the oxygen intermediates were very low (<10%). We therefore interpret 655 nm changes as reduction of the otherwise spectrally invisible CuB centre. We present a model where small anti-cooperative redox interactions occur between haem a-CuA-CuB (steady-state potential ranges: CuA, 212-258 mV; haem a, 254-281 mV; CuB, 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 a3, but the haem a potential closely matches cytochrome c at all times.

Nitric oxide can inhibit mitochondrial cytochrome oxidase in both oxygen competitive and uncompetitive modes. A previous model described these interactions assuming equilibrium binding to the reduced and oxidised enzyme respectively (Mason, et al. Proc. Natl. Acad. Sci. U S A 103 (2006) 708-713). Here we demonstrate that the equilibrium assumption is inappropriate as it requires unfeasibly high association constants for NO to the oxidised enzyme. Instead we develop a model which explicitly includes NO binding and its enzyme-bound conversion to nitrite. Removal of the nitrite complex requires electron transfer to the binuclear centre from haem a. This revised model fits the inhibition constants at any value of substrate concentration (ferrocytochrome c or oxygen). It predicts that the inhibited steady state should be a mixture of the reduced haem nitrosyl complex and the oxidized-nitrite complex. Unlike the previous model, binding to the oxidase is always proportional to the degree of inhibition of oxygen consumption. The model is consistent with data and models from a recent paper suggesting that the primary effect of NO binding to the oxidised enzyme is to convert NO to nitrite, rather than to inhibit enzyme activity (Antunes et al. Antioxid. Redox Signal. 9 (2007) 1569-1579).

Cellobiose oxidoreductase is a flavocytochrome secreted by wood-rotting fungi. The structure and functional role of the enzyme are reviewed, and a mechanism through which the enzyme produces superoxide, ferrous iron and hydrogen peroxide is proposed. The reactions of hydroxyl radicals formed by Fenton chemistry are discussed in the context of lignocellulose biodegradation.