NmerA of Tn501 mercuric ion reductase: structural modulation of the pKa values of the metal binding cysteine thiols.

To avoid nonspecific and/or undesirable binding and reactivity of metal ions with cellular components, organisms have evolved metal-specific systems for trafficking proteins. Although systems differ, those handling soft metal ions such as Hg(2+), Cu(+), Zn(2+), etc., all utilize heavy metal-associated (HMA) proteins and domains of ~70 amino acids with a conserved GMXCXXC motif in a ßαßßαß structural fold. While the conserved cysteines define a common metal binding site in these proteins, other structural features must be utilized to create metal ion, protein partner, and contextual specificities. This paper presents initial structure-function studies of the N-terminal HMA domain (NmerA) of Tn501 mercuric ion reductase (MerA) aimed at identifying structural features critical to its role in facilitating efficient transfer of Hg(2+) to the MerA catalytic core for reductive detoxification. First, NMR solution structures of reduced and Hg(2+)-bound forms of NmerA are presented that allow definition and comparison of the structure of the metal binding loop in the two states. Structural differences between the two forms are compared with differences observed in three HMA domains with different metal ion and functional contexts. Second, analyses of the UV absorbance properties of wild-type, Cys11Ala, and Cys14Ala forms of NmerA are presented that provide assignments of the pK(a) values for the two cysteine thiols of the metal binding motif. Third, results from ¹³C NMR studies with wild-type and Y62F NmerA labeled with [ß-¹³C]cysteine are presented that define a role for Tyr62 in modulating the pK(a) values of the cysteine thiols.

PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release.

Bicyclic nitroimidazoles, including PA-824, are exciting candidates for the treatment of tuberculosis. These prodrugs require intracellular activation for their biological function. We found that Rv3547 is a deazaflavin-dependent nitroreductase (Ddn) that converts PA-824 into three primary metabolites; the major one is the corresponding des-nitroimidazole (des-nitro). When derivatives of PA-824 were used, the amount of des-nitro metabolite formed was highly correlated with anaerobic killing of Mycobacterium tuberculosis (Mtb). Des-nitro metabolite formation generated reactive nitrogen species, including nitric oxide (NO), which are the major effectors of the anaerobic activity of these compounds. Furthermore, NO scavengers protected the bacilli from the lethal effects of the drug. Thus, these compounds may act as intracellular NO donors and could augment a killing mechanism intrinsic to the innate immune system.

Direct monitoring of metal ion transfer between two trafficking proteins.

To avoid the toxic effects of both essential and nonessential metal ions, cells elaborate a variety of metal ion trafficking proteins that regulate metal ion mobility. While structures of several trafficking proteins have been determined, kinetic characterization of metal ion transfer between cognate protein partners and the factors controlling transfer has lagged behind, in part due to a limitation on methods to monitor the rapid transfer. In this Communication we report studies on the kinetics of Hg2+ transfer between separately expressed components of the flavoenzyme mercuric ion reductase (MerA) that take advantage of the sensitivity of the flavin fluorescence to the charge state of a cysteine thiol in the Hg2+ binding pathway. The thiolate form of C558 in the Tn501 MerA partially quenches the fluorescence of the oxidized enzyme. Protonation or binding of Hg2+ to the thiolate increases the fluorescence, providing a sensitive probe for kinetic analysis of the Hg2+ binding reaction. The kinetics of Hg2+ transfer in both directions between the cysteine pair of the separately expressed N-terminal domain and the C-terminal cysteines (C558, C559) of the catalytic core are presented, along with a model describing the overall process.

NmerA, the metal binding domain of mercuric ion reductase, removes Hg2+ from proteins, delivers it to the catalytic core, and protects cells under glutathione-depleted conditions.

The ligand binding and catalytic properties of heavy metal ions have led to the evolution of metal ion-specific pathways for control of their intracellular trafficking and/or elimination. Small MW proteins/domains containing a GMTCXXC metal binding motif in a betaalphabetabetaalphabeta fold are common among proteins controlling the mobility of soft metal ions such as Cu(1+), Zn(2+), and Hg(2+), and the functions of several have been established. In bacterial mercuric ion reductases (MerA), which catalyze reduction of Hg(2+) to Hg(0) as a means of detoxification, one or two repeats of sequences with this fold are highly conserved as N-terminal domains (NmerA) of uncertain function. To simplify functional analysis of NmerA, we cloned and expressed the domain and catalytic core of Tn501 MerA as separate proteins. In this paper, we show Tn501 NmerA to be a stable, soluble protein that binds 1 Hg(2+)/domain and delivers it to the catalytic core at kinetically competent rates. Comparison of steady-state data for full-length versus catalytic core MerA using Hg(glutathione)(2) or Hg(thioredoxin) as substrate demonstrates that the NmerA domain does participate in acquisition and delivery of Hg(2+) to the catalytic core during the reduction catalyzed by full-length MerA, particularly when Hg(2+) is bound to a protein. Finally, comparison of growth curves for glutathione-depleted Escherichia coli expressing either catalytic core, full-length, or a combination of core plus NmerA shows an increased protection of cells against Hg(2+) in the media when NmerA is present, providing the first evidence of a functional role for this highly conserved domain.

Quantitative identification of the protonation state of histidines in vitro and in vivo.

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.