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* Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720-1460 and Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065
3 To whom correspondence should be addressed. E-mail: marletta{at}cchem.berkeley.edu.
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ABSTRACT |
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KEY WORDS: • nitric oxide • metals • diptheria toxin repressor protein • guanylate cyclase
It is now well established that nitric oxide (NO)4 occupies a central position in mammalian biochemistry (1). The initial findings were greeted with skepticism, because on the surface it seems to run against common sense that Nature would have chosen a highly toxic molecule such as NO to function as an integral paracrine signaling agent. Yet it is apparent that such a choice has been made. Clearly systems have evolved that allow NO to function in biological settings without compromising the health of the organism.
There are two principle physiological functions of NO. The first is cell-to-cell signaling, where dilation of blood vessels serves as a prototypical example. The second is the host response to infection, where NO plays an integral role in the cell-killing and -stasis chemistry that is essential to the immune system. In signaling, the central features involve a constitutive isoform of NO synthase (NOS) that is regulated by Ca2+ and calmodulin (CaM) in the generator cell and the soluble isoform of guanylate cyclase (sGC) in the target cell. In blood vessel dilation, the generator cell is the endothelium and the target tissue is smooth muscle.
The use of NO by the immune system is perhaps not a surprise. It appears that the immune system, more specifically macrophages, have harnessed the toxic properties of NO to assist in controlling infections and other invasions by foreign organisms such as tumor cell growth. In this setting, NO functions much as other small-molecule toxicants such as superoxide, hydrogen peroxide and hypochlorous acid. Generated at the site of infection, these molecules react indiscriminately with their surroundings. NO is chemically reactive (described in more detail below: see NO reactivity), and this reactivity as well as its ability to form stable complexes with some metalloproteins result in NO being quite toxic. Localized concentrations of NO in a site of inflammation approach 4–5 µmol/L and are toxic to all cells in the vicinity. The localized high concentrations of NO and the lack of a specific target differentiate NO used in the immune response from NO used in signaling.
NO in signaling
Signaling, on the other hand, represents the other extreme of using NO in a biological setting and places stringent demands on any system wishing to use it safely. Tight biosynthetic regulation of NO synthesis is the first important step, because toxicity to the cell that is synthesizing NO must be avoided (2). The regulation of activity is strictly controlled by Ca2+/CaM in the following way: i) transient increases in Ca2+ result from an external stimulus; ii) the released Ca2+ binds to CaM, which in turn binds to NOS; iii) the Ca2+/CaM/NOS complex is catalytically active and makes NO until iv) Ca2+ is reabsorbed, thereby shifting the equilibrium back to free NOS, which is now turned off. The critical switch then is the specific stimulus-induced release of Ca2+, which controls NOS activity such that nanomolar concentrations of NO are synthesized: this is a concentration low enough not to deleteriously affect the generator cell. However, NO is reactive with, among other things, molecular oxygen. Although the debate continues concerning free NO diffusion versus transport as a semistable entity, the existing data show no need for a transport system. Free diffusion of NO, for example, from the endothelium to smooth muscle, occurs with loss of NO due to decomposition en route; however, picomolar concentrations of NO have been measured in smooth muscle cells after stimulation of NOS in the endothelium.
This discussion sets the boundaries for NO signaling. NO synthesis in the generator cells is strictly controlled such that only low concentrations (nanomolar) are made to avoid toxicity to those cells. However, the amount of NO that makes it into target cells such as smooth muscle cells is on the order of picomoles. This dictates that sGC must be highly sensitive; otherwise, signal transduction with NO would never work. How does sGC accomplish this task? A heterodimeric protein, sGC is composed of 
- and ß-subunits. Additionally, it is a hemoprotein that contains a ferrous protoporphyrin IX heme prosthetic group (PPIX) that has high binding affinity for NO. Craven and DeRubertis (3–
5) and Ignarro (6) were the first to characterize a role for heme in the activation of sGC. Schultz and colleagues (7) first isolated enough of the enzyme to obtain a visible ultraviolet spectrum, which suggested that a histidine residue of the protein was ligated to the heme. Studies from this laboratory on the native enzyme (8–
15) and with a heme-binding fragment of the ß-subunit (16–
20) have more clearly defined how sGC works. Indeed, this work clearly showed that sGC contains PPIX with histidyl ligation, and that it binds NO and CO weakly and O2 not at all. Given that the heme is identical in ligation to hemoglobin and myoglobin, the fact that the protein did not bind O2 was unusual. A key molecular question was: How did sGC eliminate O2 binding? This was a critical question, because the concentration of O2 in cells would dwarf the NO concentration in target cells. The molecular engineering appears to be quite simple. Over the years, it has been shown that the affinity of O2 for a ferrous heme is positively correlated with the strength of the Fe–His bond (His being the so-called proximal ligand). In certain well-studied heme proteins (the globins, for example), the protein environment on the distal side of the porphyrin can also play an important role in O2 binding. However, in sGC, a series of studies with the heme-binding fragment showed that the extraordinarily weak Fe–His bond was the key determinant in excluding O2 binding. Without this engineering, a NO receptor using a Fe2+ porphyrin could never be used in the presence of high concentrations of O2.
Do pathogens respond to NO?
There have been several reports over the last few years that pathogens might respond to NO where that response allows them to escape the toxic properties of NO. Most studies have focused on the regulation of flavohemoglobin in Escherichia coli (21– 23). Our studies have centered on a model system that involves iron acquisition in gram-positive organisms such as Corynebacterium diphtheriae. As the name implies, the diphtheria toxin repressor protein (DtxR) was discovered through studies involving diphtheria and the diphtheria toxin (24). The conclusions reached from a series of experiments were that the toxin was carried by a phage, and upon infection of the host bacterium, the gene for the toxin fell under control of DtxR. DtxR is best characterized as an iron-dependent negative regulatory element that controls the expression of siderophores in Corynebacteria. Several other genes are known to be regulated by DtxR, and almost certainly there are more yet to be discovered.
The question we sought to answer was: Does NO deactivate DtxR, and if so, what is the mechanism of this deactivation? In summary, we have found i) using an E. coli reporter system, NO deactivates DtxR in vivo; ii) in vivo deactivation of DtxR occurs at physiologically relevant (low micromolar) NO concentrations; iii) using gel-shift and in vitro transcription assays, NO inhibits DtxR DNA-binding activity in vitro; and iv) inhibition is dependent on the NO concentration. Overall, we conclude that iron centers such as those in DtxR are subject to oxidation/deactivation in vivo. Whether organisms use DtxR or related proteins to sense NO and respond to the toxic challenge remains to be answered.
NO reactivity
NO has one unpaired electron, and the reactivity of NO is due primarily to this odd electron. Although so-called alternate redox forms (NO- and NO+) have been discussed, it is only NO that functions in signaling. The odd electron on NO kinetically allows the reaction with O2 to occur. N2O3 is an intermediate in the reaction of NO with O2. N2O3 reacts with water to yield NO2-, the stable product of solution decomposition. NO does not react directly with thiols as is often written; however, transition-metal catalysis of this reaction could be important in vivo. NO also reacts avidly with superoxide to yield peroxynitrite (HOONO), which can rearrange to NO3-, and react with protein side chains (most commonly with tyrosine to produce nitrotyrosines) or engage in peroxidelike oxidation reactions (with thiols, for example).
Biological systems have evolved to use NO for cell-to-cell signaling. The enzyme and receptor machinery involved is tightly controlled, specific and sensitive—all crucial criteria to be fulfilled. The nonspecific reactivity of NO is used by the immune system as part of the host response to infection. There are many key questions still to be answered regarding how NO kills pathogens and what, if any, response is mounted by the organism.
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FOOTNOTES |
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2 This work was supported by National Institutes of Health Grant CA 26731.
4 Abbreviations used: CaM, calmodulin; DtxR, diptheria toxin repressor protein; NO, nitric oxide; NOS, nitric oxide synthase; PPIX, protoporphyrin IX; sGC, soluble guanylate cyclase.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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