M. Claire Kennedy

Kinetic identification of a mitochondrial zinc uptake transport process in prostate cells

Prostate cells accumulate high cellular and mitochondrial concentrations of zinc, generally 3-10-fold higher than other mammalian cells. However, the mechanism of mitochondrial import and accumulation of zinc from cytosolic sources of zinc has not been established for these cells or for any mammalian cells. Since the cytosolic concentration of free Zn(2+) ions is negligible (estimates vary from 10(-9) to 10(-15) M), we postulated that loosely bound zinc-ligand complexes (Zn-Ligands) serve as zinc donor sources for mitochondrial import. Zinc chelated with citrate (Zn-Cit) is a major form of zinc in prostate and represents an important potential cytosolic source of transportable zinc into mitochondria. The mitochondrial uptake transport of zinc was studied with isolated mitochondrial preparations obtained from rat ventral prostate. The uptake rates of zinc from Zn-Ligands (citrate, aspartate, histidine, cysteine) and from ZnCl(2) (free Zn(2+)) were essentially the same. No zinc uptake occurred from either Zn-EDTA, or Zn-EGTA. Zinc uptake exhibited Michaelis-Menten kinetics and characteristics of a functional energy-independent facilitative transporter associated with the mitochondrial inner membrane. The uptake and accumulation of zinc from various Zn-Ligand preparations with logK(f) (formation constant) values less than 11 was the same as for ZnCl(2;) and was dependent upon the total zinc concentration independent of the free Zn(2+) ion concentration. Zn-Ligands with logK(f) values greater than 11 were not zinc donors. Therefore the putative zinc transporter exhibits an effective logK(f) of approximately 11 and involves a direct exchange of zinc from Zn-Ligand to transporter. The uptake of zinc by liver mitochondria exhibited transport kinetics similar to prostate mitochondria. The results demonstrate the existence of a mitochondrial zinc uptake transporter that exists for the import of zinc from cytosolic Zn-Ligands. This provides the mechanism for mitochondrial zinc accumulation from the cytosol which contains a negligible concentration of free Zn(2+). The uniquely high accumulation of mitochondrial zinc in prostate cells appears to be due to their high cytosolic level of zinc-transportable ligands, particularly Zn-Cit.

Evidence that phosphorylation of iron regulatory protein 1 at Serine 138 destabilizes the [4Fe-4S] cluster in cytosolic aconitase by enhancing 4Fe-3Fe cycling.

Iron-sulfur cluster-dependent interconversion of iron regulatory protein 1 (IRP1) between its RNA binding and cytosolic aconitase (c-acon) forms controls vertebrate iron homeostasis. Cluster removal from c-acon is thought to include oxidative demetallation as a required step, but little else is understood about the process of conversion to IRP1. In comparison with c-aconWT, Ser138 phosphomimetic mutants of c-acon contain an unstable [4Fe-4S] cluster and were used as tools to further define the pathway(s) of iron-sulfur cluster disassembly. Under anaerobic conditions cluster insertion into purified IRP1S138E and cluster loss on treatment with NO regulated aconitase and RNA binding activity over a similar range as observed for IRP1WT. However, activation of RNA binding of c-aconS138E was an order of magnitude more sensitive to NO than for c-aconWT. Consistent with this, an altered set point between RNA-binding and aconitase forms was observed for IRP1S138E when expressed in HEK cells. Active c-aconS138E could only accumulate under hypoxic conditions, suggesting enhanced cluster disassembly in normoxia. Cluster disassembly mechanisms were further probed by determining the impact of iron chelation on acon activity. Unexpectedly EDTA rapidly inhibited c-aconS138E activity without affecting c-aconWT. Additional chelator experiments suggested that cluster loss can be initiated in c-aconS138E through a spontaneous nonoxidative demetallation process. Taken together, our results support a model wherein Ser138 phosphorylation sensitizes IRP1/c-acon to decreased iron availability by allowing the [4Fe-4S]2+ cluster to cycle with [3Fe-4S]0 in the absence of cluster perturbants, indicating that regulation can be initiated merely by changes in iron availability.

Reaction of nitric oxide with iron bleomycin bound to DNA: properties of the nitrosyl adduct and its reaction with O2☆

During the ESR spectroscopic titration of nitrosyl-iron bleomycin, ONFe(II)Blm, with DNA, its metal domain undergoes a change in environment as the DNA base pair to drug ratio increases to 50 to 1. The 15NO stretching frequency of ONFe(II)Blm occurs at 1589 cm−1, similar to that for nitrosyl hemoglobin and myoglobin. Upon addition of DNA (3 base pairs per drug molecule), this vibration is substantially broadened. Injection of O2 into a solution of ONFe(II)BlmDNA converts the ESR signal of the nitrosyl species to low spin Fe(III) BlmDNA. NO is largely oxidized to NO2−. The combination of these products suggests that the initial reaction of ONFe(II)Blm with O2 generates Fe(III)Blm and peroxynitrite, O2NO−. If peroxynitrite is formed in the reaction, it does not cause detectable DNA damage. The structural integrity of a supercoiled DNA plasmid, pBR322, is not compromised and no base propenals are produced during this reaction.

The Iron Responsive Element (IRE), the Iron Regulatory Protein (IRP), and Cytosolic Aconitase

The fundamental role of iron in the maintenance of human health has been apparent for many years. The requirement for iron in growth and development of organisms from bacteria to humans arises because it is an essential component of proteins that perform redox and nonredox roles in a number of cellular functions. Given that iron is one of the most abundant, chemically versatile, and reactive elements in our environment, it is not surprising that nature has made extensive use of its properties. However, there are two significant problems regarding the use of iron in biological systems: its low solubility, particularly as Fe(III), and its toxicity when present in excess because of its ability to induce formation of damaging free radicals. Organisms have developed a variety of mechanisms to acquire and make use of iron for a large number of necessary functions while simultaneously reducing the incidence of inappropriate effects of this micronutrient on cell viability. Recent investigations of the regulation of iron homeostasis in mammals have identified two unique proteins: the iron regulatory proteins or IRPs,1 which act as central regulators of iron utilization. IRPs appear to represent the only members of the aconitase family of proteins that function in gene regulation (Frishman and Hentze 1996; Rouault et al. 1992). IRPs are cytosolic RNA binding proteins that modulate synthesis of proteins that function in the uptake, storage, and utilization of iron by binding to their mRNAs, thereby affecting their translation or stability. Posttranslational regulation of IRP function by iron and phosphorylation, with subsequent effects on iron metabolism, are topics of current inquiry to those interested in posttranscriptional gene regulation, iron-sulfur protein structure and function, and regulation of iron homeostasis.

Products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide

Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approximately 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7 state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g parallel = 2.11 and g perpendicular = 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approximately 2.04 species and in the case of mitochondrial aconitase, a transient signal at g approximately 2. 032 appeared. This signal is tentatively assigned to the d9 form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.