Showing papers in "Essays in Biochemistry in 1999"

Coenzyme B12 (cobalamin)-dependent enzymes.

Abstract: The B12 or cobalamin coenzymes are complex macrocycles whose reactivity is associated with a unique cobalt-carbon bond. The two biologically active forms are MeCbl and AdoCbl and their closely related cobamide forms. MeCbl participates as the intermediate carrier of activated methyl groups. During the catalytic cycle the coenzyme shuttles between MeCbl and the highly nucleophilic cob(I)alamin form. Examples of MeCbl-dependent enzymes include methionine synthase and Me-H4-MPT: coenzyme M methyl transferase. AdoCbl functions as a source of carbon-based free radicals that are unmasked by homolysis of the coenzyme's cobalt-carbon bond. The free radicals are subsequently used to remove non-acid hydrogen atoms from substrates to facilitate a variety of reactions involving cleavage of carbon-carbon, carbon-oxygen and carbon-nitrogen bonds. Most reactions involve 1,2 migrations of hydroxy-, amino- and carbon-containing groups, but there is also one class of ribonucleotide reductases that uses AdoCbl. The structures of two cobalamin-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase, have been solved. In both cases the cobalt is co-ordinated by a histidine ligand from the protein. The significance of this binding motif is presently unclear since in other cobalamin-dependent enzymes spectroscopic evidence suggests that the coenzyme's nucleotide 'tail' remains co-ordinated to cobalt when bound to the protein.

Haem iron-containing peroxidases.

Abstract: Peroxidases are enzymes that utilize hydrogen peroxide to oxidize substrates. A histidine residue on the proximal side of the haem iron ligates most peroxidases. The various oxidation states and ligand complexes have been spectroscopically characterized. HRP-I is two oxidation states above ferric HRP. It contains an oxoferryl (= oxyferryl) iron with a pi-radical cation that resides on the haem. HRP-II is one oxidation state above ferric HRP and contains an oxoferryl iron. HRP-III is equivalent to the oxyferrous state. Only compounds I and II are part of the peroxidase reaction cycle. CCP-ES contains an oxoferryl iron but the radical cation resides on the Trp-191 residue and not on the haem. CPO is the only known peroxidase that is ligated by a cysteine residue rather than a histidine residue, on the proximal side of the haem iron. CPO is a more versatile enzyme, catalysing numerous types of reaction: peroxidase, catalase and halogenation reactions. The various CPO species are less stable than other peroxidase species and more elusive, thus needing further characterization. The roles of the amino acid residues on the proximal and distal sides of the haem need more investigation to further decipher their specific roles. Haem proteins, especially peroxidases, are structure-function-specific.

Bacterial detoxification of Hg(II) and organomercurials.

Abstract: The most common bacterial mechanism for resistance to mercuric-ion species involves intracellular reduction of Hg(II) to Hg(0). Key proteins of the pathway typically include: MerR, which regulates pathway expression; MerP, which protects the external environment; MerT or MerC, which transport Hg(II) species across the inner membrane; MerA, which catalyses reduction of Hg(II); and sometimes MerB, which catalyses cleavage of C-Hg bonds in organomercurials. Cysteine residues of varying number are arranged in each of the key proteins to optimize their unique roles in sensing (high affinity), transporting (exchangeability), and reducing (redox accessibility) Hg(II). Nature's regulator of this pathway, MerR, is an exquisitely sensitive, Hg(II)-binding, DNA-binding protein that holds the system primed for immediate transcription at the slightest influx of Hg(II).

Oxygen-carrying proteins: three solutions to a common problem.

Abstract: Nature has used transition-metal ions with unpaired d-electrons to overcome the kinetic inertness of O2 and to control its thermodynamic tendency towards reduction. High-resolution X-ray crystal structures of O2-carrying proteins show that Nature has devised three distinct solutions to the problem of reversible O2 binding. The three types can be classified according to their active sites: Hb (haem iron); Hr (non-haem di-iron); and Hcy (dicopper). The reversible O2 binding to the three types of active site are formally oxidative additions: Fe(II) to Fe(III)-O2- for Hb; [Fe(II),Fe(II)] to [Fe(III),Fe(III)O(2)2-] for Hr; and [Cu(I),Cu(I)] to [Cu(II)(mu-O(2)2-) Cu(II)] for Hcy. In all cases the O-O bond is weakened, but not cleaved, upon binding. The 'textbook' explanation for discrimination against CO and O2 binding to Hb has been revised: steric constraints to the preferred linear Fe-C-O geometry imposed by the 'distal' histidine are no longer thought to play a major role. Instead, recent experimental evidence indicates that the polarity of the binding pocket favours the polar Fe-O-O unit over the relatively non-polar Fe-C-O unit, and that a C-O-binding pocket near the haem also inhibits the preferred linear Fe-C-O geometry. Reversible O2 binding to the di-iron site of Hr involves an internal proton transfer as well as electron transfer to O2, but the elementary steps governing the rates of O2 binding and release, especially the effects of the surrounding protein, remain to be delineated. An unusual side-on-bonded O2 that bridges the two copper ions explains both the unusually low O-O stretching frequency and the diamagnetism of oxyHcy. O2-activating-enzyme counterparts exist for each of the three known types of O2-carrying protein. Detailed comparisons of these protein/enzyme pairs are likely to clarify the factors that tune the delicate balance between reversible O2 binding and controlled O-O bond cleavage.

The role of efflux in bacterial resistance to soft metals and metalloids

Abstract: Bacteria have evolved various types of resistance mechanism to toxic soft metals and metalloids, including cadmium/zinc, copper/silver and arsenic/antimony. Active efflux of the metal is a frequently utilized stratagem to produce resistance by lowering the intracellular concentration to subtoxic levels. Reduction to a less-toxic form or to a form recognized by an efflux system also occurs. Pumps utilized for resistance may have evolved from normal cellular systems. For example, plasmid-mediated cadmium resistances may have evolved from a common ancestor of the pump involved in zinc homoeostasis. Pumps are more efficient than carriers and may have evolved by developing carriers that associate with ATPase subunits.

Nature's universal oxygenases: the cytochromes P450.

Abstract: Cytochromes P450 are utilized in an enormous diversity of biological reactions, including degradation of xenobiotics, generation of hormones and biosynthesis of a variety of important biological compounds. The cytochrome P450 family is a major participant in nearly all metabolism of pharmaceutical reagents. The presence of different P450 enzymes in various quantities in individuals makes the prediction of drug responses in patients highly complex. A large literature describing mechanistic studies has characterized several intermediates in the oxygenation pathway. It has recently been shown that two or more possible oxygenated forms of the P450 haem can participate in various oxygenations, with some intermediates being highly electrophilic and others being nucleophilic.

Oxygen reactions of the copper oxidases.

Abstract: The copper oxidases are a remarkable family of metalloenzymes that have evolved specialized mechanisms to accomplish the controlled reduction of dioxygen, delivering the equivalent of H2 from organic substrates to O2 to form hydrogen peroxide, a ubiquitous oxygen metabolite that is involved in a wide range of biological interactions. These enzymes display their virtuosity in dioxygen chemistry by harnessing the oxidizing power of that molecule not only during catalytic turnover, but also in transforming themselves in the biogenesis of their catalytic redox cofactor.

Biological electron-transfer reactions.

Abstract: A wide range of biological processes makes extensive use of electron-transfer reactions. Rigorous characterization of a biological electron-transfer reaction requires a combination of kinetic, thermodynamic, structural and theoretical methods. The rate of electron transfer from an electron donor to an electron acceptor through a protein is dependent on the difference in reduction potential of the electron acceptor and electron donor and the distance over which electron transfer occurs. The manner in which the rate of electron transfer also depends on the structure of the protein located between the electron donor and acceptor sites remains an active topic of investigation. Diverse protein-engineering strategies are providing new insights into fundamental mechanistic considerations regarding electron-transfer properties of biological molecules, and they can provide novel means by which insights concerning biological electron-transfer reactions can be employed to develop new and useful types of chemistry.

Non-haem iron-containing oxygenases involved in the microbial biodegradation of aromatic hydrocarbons.

Abstract: A wide variety of aromatic hydrocarbons can be degraded aerobically by micro-organisms. A large fraction of the metabolic pathways are initiated by oxygenases containing Fe(II) at the active sites, which participates in the oxygenation and activation of the hydrocarbons. Mono-oxygenations and dioxygenations are found in these pathways. Some of these enzymes can catalyse either or both reactions, depending on the nature of the substrate. Two general themes are found: mononuclear Fe(II) centres that must be reduced by one electron at a time, or di-iron centres that can be reduced by two electrons. The electrons from NAD(P)H can be delivered by either an electron-transfer chain consisting of a flavin and one or more [2Fe-2S] centres, or a pterin. Proposed mechanisms generally involve higher oxidation states of the iron (Fe = O), analogous to those for P450, and peroxidase systems. These strong oxidants are necessary to oxidize aromatic and aliphatic compounds. Mechanisms currently considered viable for these reactions require significant changes in ligation during catalysis. The structures of the non-haem iron centres may be particularly well-suited for such transformations.