Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Bacteriocins of gram-positive bacteria.

The heme oxygenases, which consist of constitutive and inducible isozymes (HO-1, HO-2), catalyze the rate-limiting step in the metabolic conversion of heme to the bile pigments (i.e., biliverdin and bilirubin) and thus constitute a major intracellular source of iron and carbon monoxide (CO). In recent years, endogenously produced CO has been shown to possess intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. The era of gaseous molecules in biomedical research and human diseases initiated with the discovery that the endothelial cell-derived relaxing factor was identical to the gaseous molecule nitric oxide (NO). The discovery that endogenously produced gaseous molecules such as NO and now CO can impart potent physiological and biological effector functions truly represented a paradigm shift and unraveled new avenues of intense investigations. This review covers the molecular and biochemical characterization of HOs, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by environmental stress. Furthermore, the current understanding of the functional significance of HO shall be discussed from the perspective of each of the metabolic by-products, with a special emphasis on CO. Finally, this presentation aspires to lay a foundation for potential future clinical applications of these systems.

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Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications.

THE OCCURRENCE AND DETERMINATION OF δ-AMINOLEVULINIC ACID AND PORPHOBILINOGEN IN URINE

Publisher Summary This chapter deals with the absorption spectra of proteins and amino acids The colored proteins are conjugated proteins in which the protein carrier is colorless This transparency of protein solutions extends into the ultraviolet region of the spectrum and many proteins do not absorb radiation of longer wavelength than 2500 Ǻ The essential protein fabric, consisting of a peptide chain in various forms, is not responsible for absorption at longer wavelengths In case of fibrous proteins, there is some evidence that the peptide fabric is responsible for absorption in this region Many proteins absorb in this region This absorption is due to the aromatic amino-acids present in the protein The advent of quantitative methods of spectrophotometry is the basis of a method of determining tyrosine and tryptophan in proteins The striking property of proteins is their transparency, indicating a high degree of electronic saturation The configurational stability of the protein molecule depends entirely on extra-valence forces and not on unsaturation, which would result in high absorption in the ultraviolet The absence of such rigidifying bonds endows the protein with its unique characters of plasticity, while the number-sequence of side chains gives its chemical constancy These two properties allow these molecules to be arranged in large polymorphic masses to form a matrix fabric of recurrent pattern in media, which are essentially aqueous The material reviewed is principally derived from the study of homogeneous absorbing systems, in which the inhomogeneity is finer in grade by several orders than the dimensions of the exploring light beam

Ultraviolet absorption spectra of proteins and amino acids.

An in-depth study of any metabolic pathway not only involves the chemical transformations that occur in both synthetic and degradative steps but must consider as well detailed mechanisms of each step, control mechanisms which may be involved at one or several particular places, genetic differences in different cells, and the influence the particular set of reactions under study has on the function of the whole cell. In a study of porphyrin, heme, vitamin B12, and chlorophyll synthesis one is a t least concerned with the problems generally outlined above. At present, the precursors of the porphyrins and related compounds and the general outline of their synthesis are known along with some of the mechanisms of some of the early steps. Other details, such as the mechanism of formation of the functional isomer of porphyrins from the pyrrole, porphobilinogen, the control of porphyrins and heme synthesis, and possible genetic differences in the mechanisms of some of the enzymes involved are at present not known. An approach t o the particular problems concerned with porphyrin synthesis, which is indeed not unique t o this system, is t o study in detail the architecture of enzymes involved in porphyrin synthesis and, of course, t o use a variety of other methods along with detailed protein studies; these may aid in the elucidation of some of the particular problems and perhaps even throw some light on the basic understanding of enzymic catalysis in general. The following summary will deal with some of our more recent efforts in this area and some of our projected experiments whose design arose from our findings. Since the data for some of the conclusions stated here have in some instances been published or are about to be published with my coworkers I shall not repeat the experimental data but will only give the conclusions in this communication.

Porphyrin synthesis: some particular approaches.

The viscosity of Rous sarcoma extracts can be reduced with a polysaccharide enzyme isolated from pneumococcus or testis without destruction of the active agent and thus more concentrated active filtrates can be obtained. The active agent can be precipitated with basic proteins. The basic protein can be separated from the components originally coming from the tumor filtrate by electrophoresis.

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Studies of the transmissible agent of the rous chicken sarcoma i : precipitation with basic proteins.

The relationship of serine to porphyrin synthesis

Publisher Summary This chapter discusses the enzymatic control of pyrrole synthesis (porphobilinogen). It focuses on the mechanism of the reaction and the nature and conformation of the enzyme involved in a particular biological system. Porphobilinogen is enzymatically synthesized (δ-aminolevulinic acid dehydratase) from two molecules of δ-aminolevulinic acid that arise from the condensation of succinyl-CoA and glycine (δ-aminolevulinic acid synthetase). The synthesis of the aminoketone, δ-aminolevulinic acid, can be considered to be the first committed step in the series of reactions that lead to the synthesis of porphyrins and related compounds, whereas the synthesis of the pyrrole is the step in which aromatization occurs. The latter reaction may also be considered a distinct committed step, because an aliphatic compound is changed into an aromatic ring compound. If these two enzymatic reactions are committed steps, they may both be subjected to metabolic control by feedback inhibition and/or by repression.

David Shemin

Papers

Porphyrin synthesis: some particular approaches.

Studies of the transmissible agent of the rous chicken sarcoma i : precipitation with basic proteins.

The relationship of serine to porphyrin synthesis

Mechanism and Control of Pyrrole Synthesis

The Succinate‐Glycine Cycle; The Rôle of δ‐Aminolevulinic Acid in Porphyrin Synthesis