Patrick J. Farmer

The reduction potential of nitric oxide (NO) and its importance to NO biochemistry

A potential of about −0.8 (±0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion (3NO−) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to 1NO− is highly unfavorable, with a predicted reduction potential of about −1.7 (±0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and −0.35 V for the NO/3NO− and NO/1NO− couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of 3NO− has been reevaluated as 11.6 (±3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, 1NO−, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O2 under reductive conditions is discussed.

Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe2S2] ferredoxins, products of PETF, FDX2–FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP+ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (Em = −321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.

Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions.

Melanins are colloidal pigments known to have a high affinity for metal ions. In this work, the nature of the metal-binding sites are determined and the binding affinities are quantified. Initial potentiometric titrations have been performed on synthetic dihydroxyindole (DHI) melanin solutions to determine the chemical speciation of quinole/quinone subunits. Two types of acidic functionalities are assignable: catechol groups, with pK(a) between 9 and 13, and quinone imines (QI), with pK(a) of 6.3. The presence of the quinone-imine tautomer has, to our knowledge, never been assessed in polymeric melanins. Melanin solutions obtained from N-methylated DHI lack the pK(a) 6.3 buffer, consistent with its inability to form the quinone-imine tautomer. EPR spectroscopy of the DHI-melanin samples demonstrates that the semiquinone radical is in too low a concentration to contribute to the bulk binding of metals. Changes in the titration curves after addition of Cu(II) and Zn(II) ions were analyzed to obtain the binding constants and stoichiometry of the metal-melanin complexes, using the BEST7 program. UV-Vis spectra at neutral and high pH are used to identify absorbances due to Cu-bound quinone imine and catechol groups. The derived binding constants were used to determine speciation of the Cu(II) and Zn(II) ions coordinated to the quinone imine and catechol groups at various pH. The mixed complexes, Zn(QI)(Cat)(-) and Cu(QI)(Cat)(-) are shown to dominate at physiological pH.

The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.

Etiologic Pathogenesis of Melanoma: A Unifying Hypothesis for the Missing Attributable Risk

Melanoma is the eighth most common malignancy in the United States and has shown rapid increases in its incidence rate over the past two decades, especially in early-stage disease [(1][1], [2][2], [3][3], [4)][4] . A recent analysis of data from the Surveillance Epidemiology and End Results Program

Redox behavior of melanins: direct electrochemistry of dihydroxyindole-melanin and its Cu and Zn adducts

Synthetic melanin films, formed on electrode surfaces by oxidative polymerization of 5,6-dihydroxyindole solution, were used to directly measure the chromophores redox reactivity. Films on optically transparent indium-tin oxide (ITO) electrodes allow correlation of spectral changes with electrochemical potential. Spectroelectrochemical titrations show an initial reversible transformation that is ascribed to formation of a unique quinone-imine chromophore. The apparent E(1/2) for maximum quinone-imine formation is approximately 125 mV (vs. Ag/AgCl) but at potentials higher than 100 mV, an irreversible bleaching is evident. Correlation of the current with the monomer concentration implies that only one in six monomers is oxidized to the quinone-imine before the irreversible bleaching occurs. Films pretreated with CuCl(2) and Zn(CH(3)COO)(2) show elevated quinone-imine absorbances, even under reducing conditions, indicating a preferential stabilization of this state by coordination to the metals.

Reactions of HNO with heme proteins: new routes to HNO-heme complexes and insight into physiological effects

The formation and interconversion of nitrogen oxides has been of interest in numerous contexts for decades. Early studies focused on gas-phase reactions, particularly with regard to industrial and atmospheric environments, and on nitrogen fixation. Additionally, investigation of the coordination chemistry of nitric oxide (NO) with hemoglobin dates back nearly a century. With the discovery in the early 1980s that NO is biosynthesized as a molecular signaling agent, the literature has been focused on the biological effects of nitrogen oxides, but the original concerns remain relevant. For instance, hemoglobin has long been known to react with nitrite, but this reductase activity has recently been considered to be important to produce NO under hypoxic conditions. The association of nitrosyl hydride (HNO; also commonly referred to as nitroxyl) with heme proteins can also produce NO by reductive nitrosylation. Furthermore, HNO is considered to be an intermediate in bacterial denitrification, but conclusive identification has been elusive. The authors of this article have approached the bioinorganic chemistry of HNO from different perspectives, which have converged because heme proteins are important biological targets of HNO.

Melanosomal Damage in Normal Human Melanocytes Induced by UVB and Metal Uptake—A Basis for the Pro‐oxidant State of Melanoma†

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro‐oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)‐treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single‐dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.

Nitrosyl hydride (HNO) as an O2 analogue: long-lived HNO-adducts of ferrous globins

Nitrosyl hydride, HNO or nitroxyl, is the one-electron reduced and protonated form of nitric oxide. HNO is isoelectronic to singlet O(2), and we have previously reported that deoxymyoglobin traps free HNO to form a stable adduct. In this report, we demonstrate that oxygen-binding hemoglobins from human, soy, and clam also trap HNO to form adducts which are stable over a period of weeks. The same species can be formed in higher yields by careful reduction of the ferrous nitrosyl adducts of the proteins. Like the analogous O(2)-Fe(II) adducts, the HNO adducts are diamagnetic, but with a characteristic HNO resonance in (1)H NMR at ca. 15 ppm that splits into doublets for H(15)NO adducts. The (1)H and (15)N NMR resonances, obtained by HSQC experiments, are shown to differentiate subunits and isoforms of proteins within mixtures. An apparent difference in the reduction rates of the NO adducts of the two subunits of human hemoglobin allows assignment of two distinct nitrosyl hydride peaks by a combination of UV-vis, NMR, and EPR analysis. The two peaks of the HNO-hHb adduct have a persistent 3:1 ratio during trapping reactions, demonstrating a kinetic difference between HNO binding at the two subunits. These results show NMR characterization of ferrous HNO adducts as a unique tool sensitive to structural changes within the oxygen-binding cavity, which may be of use in defining modes of oxygen binding in other heme proteins and enzymes.

The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex THEORETICAL AND EXPERIMENTAL STUDIES

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 Å. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp38 and Trp106, and at the edge of the protein-protein interface, Tyr33 and Arg66, were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Δ106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Δ106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp106 and Tyr33/Arg66 substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr33, Arg66, and Trp106 prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106 but are likely to include Asp38 and Cys39.