Maria G. Mason

Cytochrome bd confers nitric oxide resistance to Escherichia coli

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.

Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity

ABSTRACT 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 actinomyceteStreptomyces 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 non-iron-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.

Rotting by radicals - the role of cellobiose oxidoreductase?

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.

A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase.

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).

The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres.

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 enzymes 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.

Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues.

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.

Oxygen reduction by cellobiose oxidoreductase: the role of the haem group.

We have used optical and electron paramagnetic spectroscopy to study the flavohaem enzyme cellobiose oxidoreductase (CBOR) from Phanerochaete chrysosporium. We have examined redox cycles of the enzyme in which the oxidation of cellobiose to cellobionolactone is coupled to the reduction of oxygen. During turnover flavin can reduce oxygen with one electron to produce superoxide or two electrons to produce hydrogen peroxide. Addition of superoxide dismutase significantly extended the time courses of these cycles, slowing the re‐oxidation rate of both cofactors. Addition of catalase also affected the haem time course, but to a lesser extent. Experiments in which superoxide was generated in the reaction mixture showed that this radical greatly enhanced the rate of haem re‐oxidation. From these results we propose a mechanism in which reactive oxygen species generation by CBOR flavin subsequently re‐oxidises CBOR haem. We discuss this mechanism in relationship to the biological function of this enzyme, namely lignocellulose degradation.

Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen.

Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non‐covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped‐flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033–21040 [1]). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH. Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH−). Reaction of this with dioxygen leads to transient radical (FADH) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.

Steady State Redox Levels in Cytochrome Oxidase: Relevance for in Vivo Near Infrared Spectroscopy (Nirs)

In the visible/NIR (600-900 nm) three different redox centres are potentially detectable in vivo in mitochondrial cytochrome c oxidase: haem a (605nm), the binuclear haem a3/CuB centre (655 nm) and CuA (830 nm). In this paper we report changes in the steady state reduction of these centres following increases in the rate of electron entry into the purified enzyme complex under conditions of saturating oxygen tension. As turnover is increased all three centres becomes progressively reduced. Analysis of the steady states indicated that all three centres remained in apparent equilibrium with cytochrome c throughout the titration. The calculated redox potentials of CuA (+224 mV) and haem a (+267 mV) were consistent with previous equilibrium data. The 655 nm band was also found to be oxygen and flux sensitive. It may be a useful additional in vivo detectable chromophore. However, it titrated with an apparent redox potential of +230 mV, far from its equilibrium value (+400 mV). The implications of these results for the interpretation of non invasive measurements of mitochondrial function are discussed.

A quantitative approach to nitric oxide inhibition of terminal oxidases of the respiratory chain.

Inhibition of terminal respiratory oxidases by nitric oxide (NO) plays important physiological roles in signaling and host defense. Using a bacterial quinol oxidase and mitochondrial cytochrome c oxidase, this chapter describes simple polarographic methods to quantify the kinetic characteristics of inhibition by NO. This chapter points out the inherent pitfalls of both experimental design and data analysis and compares alternative methods. Additionally, it describes a system designed to acquire polarographic and spectral data simultaneously to permit identification of spectral intermediates under defined conditions.