Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIF⌬1-120) exhibit NADH oxidase activity, whereas superoxide anion (O 2 ؊ ) is formed. AIF⌬1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIF⌬1-120, which lacks FAD, has no NADH oxidase activity. However, native AIF⌬1-120, apoAIF⌬1-120, and the reconstituted (FAD-containing) holoAIF⌬1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIF⌬1-120 assessed on purified nuclei in a cellfree system. Conversely, blockade of the apoptogenic function of AIF⌬1-120 with the thiol reagent parachloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.

Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysRtype (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators.Macrophages are important weapons of host innate immunity, exhibiting a panoply of concerted strategies for microbial elimination. These strategies include the creation of an environment hostile to bacteria proliferation caused by, among other factors, the production of small diffusible reactive molecules such as nitric oxide (NO). 1 The NO released by eukaryotes is a product of the enzymatic oxidation of L-arginine by NO synthases, and it regulates a plethora of important processes such as signaling, neuronal communication, vasodilatation, smooth muscle relaxation, and inhibition of platelet aggregation. However, these functions are achieved by using low amounts of NO and, once the concentration of NO rises above micromolar levels, the molecule becomes harmful and causes serious deleterious effects, namely tissue inflammation, chronic infection, malignant transformations, and degenerative diseases. Nevertheless, high concentrations of NO are used to fight invading prokaryotic pathogens and parasites. The NO released by macrophages is not the only source of NO that microbes need to deal with, because this compound is also produced abiotically (e.g. by decomposition of nitrite) and biotically by denitrifiers/ammonifiers or other bacteria (1).Like all living organisms, bacteria have the ability to respond to aggression by developing a series of not yet fully understood mechanisms that include damage repair, eliciting the SOS system, resistance increase, and use of virulence as a counteracting tactic. Apart from the microbial membranebound heme-iron NO reductases, the cytoplasmatic globins, the flavodiiron NO reductases, and the multiheme nitrite reductases (2) are also proposed to metabolize NO. Escherichia coli contains these three proteins: (i) the flavorubredoxin NorV, a flavodiiron NO reductase (3), the discovery of which allowed the identification of similar enzymes in a large number of prokaryotic and protozoan genomes (4); (ii) the flavohemoglobin HmpA (5)...

The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.

Carbon monoxide (CO) is endogenously produced in the human body, mainly from the oxidation of heme catalyzed by heme oxygenase (HO) enzymes. The induction of HO and the consequent increase in CO production play important physiological roles in vasorelaxation and neurotransmission and in the immune system. The exogenous administration of CO gas and CO-releasing molecules (CO-RMs) has been shown to induce vascular effects and to alleviate hypoxia-reoxygenation injury of mammalian cells. In particular, due to its anti-inflammatory, antiapoptotic, and antiproliferative properties, CO inhibits ischemic-reperfusion injury and provides potent cytoprotective effects during organ and cell transplantation. In spite of these findings regarding the physiology and biology of mammals, nothing is known about the action of CO on bacteria. In the present work, we examined the effect of CO on bacterial cell proliferation. Cell growth experiments showed that CO caused the rapid death of the two pathogenic bacteria tested, Escherichia coli and Staphylococcus aureus, particularly when delivered through organometallic CO-RMs. Of importance is the observation that the effectiveness of the CO-RMs was greater in near-anaerobic environments, as many pathogens are anaerobic organisms and pathogen colonization occurs in environments with low oxygen concentrations. Our results constitute the first evidence that CO can be utilized as an antimicrobial agent. We anticipate our results to be the starting point for the development of novel types of therapeutic drugs designed to combat antibioticresistant pathogens, which are widespread and presently a major public health concern.Carbon monoxide (CO) is a colorless and odorless diatomic gas, chemically inert, that occurs in nature as a product of oxidation or combustion of organic matter. Owing to its lethal effect when present in high concentrations, CO was considered for many years to be only an environmental toxicant that results from air pollution by automobile exhaust. The knowledge that the human body is able to produce small quantities of CO and the evidence that CO derived from heme oxygenase activity contributes to important intracellular functions have modified our perception of CO as a pernicious toxin to include its beneficial effects (15,16,22). In consequence, the application of CO gas or CO-releasing molecules (CO-RMs) has emerged as a new therapeutic strategy in medicine (10,13,18). The evolution of CO from a toxicant to a molecule of mounting importance in mammals finds a parallel in another diatomic molecule, nitric oxide (NO) (17). NO is produced in the body by the nitric oxide synthase and shares with CO many downstream signaling pathways and regulatory functions, in particular, those associated with the activation of soluble guanylyl cyclase (7,8,12). In addition, there is an interplay between the two molecules, since it is proposed that CO is a modulator of nitric oxide synthase (10, 22) and NO up-regulates heme oxygenase (19,20), which in turn catalyzes the oxidative degrad...