ion step follows the decarboxylation, which is consistent with the deuterium isotopic effects observed for thymine 7-hydroxylase which indicate that an irreversible step (or steps) occurs prior to the C-H bond breaking.395 It has also been shown for prolyl 4-hydroxylase that a substrate-derived radical is generated in the reaction, which is consistent with a rebound mechanism.437 It is important to point out that no oxygen intermediate (i.e., bridged superoxo or oxo-ferryl) has been observed for any R-KGdependent enzyme. This warrants future theoretical and experimental study. A detailed molecular mechanism has been proposed for IPNS based on spectroscopic and crystallographic studies.422 Resting IPNS/FeII is also 6C and thus relatively stable toward dioxygen. Substrate ACV binds directly to FeII IPNS through its thiolate group, providing an open coordination position at the FeII. O2 can then react to form an FeIII-superoxo intermediate. This intermediate is suggested422 to perform the first hydrogen-atom abstraction step and close the â-lactam ring, resulting in the formation of the first water molecule and generating an FeIVdO-II intermediate, which completes the second ringclosure process by hydrogen-atom abstraction forming a thiazolidine ring. Previously proposed mechanisms of ACCO involved direct binding of cosubstrate ascorbate to the iron before O2 as part of the oxygen activation process.438,439 The EPR and ESEEM studies of the NO complex of ACCO suggested a quite different molecular mechanism for ACCO.435 An FeIII-superoxo intermediate is proposed. Whether it is preceded by a 6C f 5C process with substrate binding is presently under study.440 This intermediate is thought to initiate a radical process by single hydrogen-atom abstraction or electron-coupled proton transfer (PT)ion or electron-coupled proton transfer (PT) from the bound amino group. The resulting substrate radical may undergo spontaneous conversion into products. The role of cosubstrate ascorbate is proposed to reduce the toxic peroxo byproduct to water. Alternatively, the two-electron reduction of FeIIIsuperoxo by the cosubstrate ascorbate could result in an FeIVdO-II intermediate which initiates the radical reaction.435 4. Rieske-Type Dioxygenases Biochemical Characterization. The Rieske ironsulfur center is a two iron-two sulfur cluster ([2Fe2S]) which has a 2His (on one iron), 2Cys (on the other iron) coordination environment, instead of the 4Cys present in plant ferredoxins. It plays a key role in the electron transport pathway in membranebound cytochrome complexes as well as in some dioxygenases.441 The latter are mainly comprised of two protein components: a reductase containing flavin and a ferredoxin [2Fe-2S], and a terminal oxygenase containing a Rieske [2Fe-2S] cluster and a non-heme iron active site.442 Except for the recently reported alkene monooxygenase that has a binuclear iron site in its terminal oxygenase,10 most of the Rieske-type oxygenases have a mononuclear iron site, which is believed to be the site of dioxygen activation and substrate oxygenation.442,443 The majority of the Rieske-type mononuclear non-heme oxygenases form a family of enzymes which are aromatic-ring-hydroxylating dioxygenases. These catalyze the regioand stereospecific cis-dihydroxylation of an aromatic ring using dioxygen and NAD(P)H (Table 1). Examples include benzene dioxygenase (BDO, EC 1.14.12.3),444 phthalate dioxygenase (PDO, EC 1.14.12.7),445 toluene dioxygenase (EC 1.14.12.11),446 and naphthalene 1,2-dioxygenase (NDO, EC 1.14.12.12),447 which initiate the aerobic degradation of aromatic compounds in the soil bacteria and are targets for bioengineering in bioremediation. This step is the first step in the pathway that ultimately leads to ring cleavage by the intraand extradiol dioxygenases (sections II.B.2 and II.C.1).443 Besides these bacterial dioxygenases, other Rieske-type mononuclear non-heme oxygenases include anthranilate 1,2-dioxygenase (EC 1.14.12.1),448 which deaminates and decarboxylates the substrate to produce catechol; chlorophenylacetate 3,4-dioxygenase (EC 1.14.2.13),449 which converts substrate to catechol with chloride elimination; and 4-methoxybenzoate O-demethylase (putidamonooxin),450 which catalyzes the conversion of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and formaldehyde. The reductase component is usually a monomer (MW ) 12-15 kDa) and utilizes flavin to mediate ET from the two-electron donor NAD(P)H to the oneelectron acceptor [2Fe-2S] cluster and is specific to each terminal oxygenase; other electron donors do not support efficient oxygenation.442 The crystal structure of phthalate dioxygenase reductase is available.451 The terminal oxygenases are large protein aggregates (MW ) 150-200 kDa) containing either multiples of R subunits (BDO R2, PDO R4) or an equimolar combination of R and â subunits (toluene dioxygenase R2â2, NDO R3â3). The R subunits contain a Rieske [2Fe-2S] cluster and a catalytic non-heme FeII center. â subunits do not seem to be involved in the catalytic function (vide infra). Kinetics. Steady-state kinetic studies coupled with various rapid reaction studies of the partial reactions of PDO allowed Ballou et al. to propose a kinetic scheme (Scheme 15).443 On the basis of steady state 278 Chemical Reviews, 2000, Vol. 100, No. 1 Solomon et al.

Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of alpha 2 beta gamma delta (refs 1-3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4-6). We have recently elucidated the complete primary structures of the alpha-, beta- and delta-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides. Here, we report the whole primary structure of the gamma-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.

Structural homology of Torpedo californica acetylcholine receptor subunits

Dioxygen Activation by Enzymes with Mononuclear Non-Heme Iron Active Sites

Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis.

Publisher Summary The chapter discusses the motions in proteins. Gelation of gelatin or precipitation of casein with mild acid are examples of reversible changes in which structural motility of the protein chains or subunits is recognized as playing a part. The sometimes reversible spreading of monolayers derived from mondisperse proteins provides some of the first evidence for the true plasticity of protein molecules and paved the way for the elegant demonstration of reversible conformational transitions. The tracing of the passage of an unfolded polypeptide chain to the compact, native structure is a primary concern of protein chemistry. One segment of polypeptide chain adapts its backbone and sidechain conformation to the structure of another segment in the process of forming the compact, functional structure of a protein was made particularly vivid by experiments on the reassociation of cleaved fragments. Overall translational and rotational motion is observed with a protein in solution, apart from any intramolecular motions. These overall motions are those that remain if the protein molecule is treated as a rigid matrix. Rotational reorientation of intramolecular components involves rotations about individual interatomic bonds. Concerted motions in proteins involve the movement of groups of side chains or segments of backbone relative to other structures. Motions of interacting ions and small molecules produce fluctuations in protein structure in addition to Brownian transfer of momentum. Time scales of various types of motions are important both for experimental recognition of the time-dependent processes and for analyzing their importance in the functional molecular mechanics. The chapter also discusses the dominant methods of observation that provide information about frequencies and amplitudes of motions in proteins.

Motions in proteins.

1
The conversion of the native iron-containing lipoxygenase-1 into yellow and purple ferric enzyme species by 13-L-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (R-13-OOH) was studied by measuring the absorbance changes at 330 nm and 580 nm, respectively in the stopped-flow apparatus. The pseudo-first-order rate constant for this reaction was found to be 50 s−1 at 4.4°C, either in the presence or absence of O2. This rate constant is lower than the one for R-13-OOH formation from linoleic acid and O2 catalysed by lipoxygenase-1: kcat= 232 s−1 at 4.4°C.

2
The coloured ferric enzymes are formed only in the presence of R-13-OOH; therefore the formation of the coloured ferric enzymes from oxygen, linoleic acid and the native enzyme indicates that the latter is active even before it is converted into the ferric forms.

3
Under anaerobic conditions the yellow and purple enzyme species are rapidly converted into colourless ferrous enzyme species by linoleic acid at a rate which is faster than the formation of the coloured enzymes from R-13-OOH and the native ferrous enzyme.

4
Both the aerobic formation of the coloured ferric enzymes and the anaerobic bleaching of these enzymes occur more slowly when [11-2H2]linoleic acid instead of unlabeled linoleic acid is used. The kinetic deuterium isotope effect as estimated from the rates of aerobic formation of R-13-OOH is approximately 9, whereas in the anaerobic conversion of R-13-OOH and deuterated linoleic acid a value of 1.1 is found. This demonstrates that H abstraction from linoleic acid determines the rate of the aerobic formation but not of the anaerobic conversion of R-13-OOH.

5
The rate constant for the anaerobic conversion of [11-2H2]linoleic acid by the yellow ferric enzyme species, k(2H) = 30 s−1 at 25°C, is close to the rate constant for the overall oxygenation reaction of the deuterated substrate, kcat(2H) = 32 s−1. For unlabelled linoleic acid the rate constants of the anaerobic H-abstraction step and the overall oxygenation reaction were found to be 111 s-1 and 290 s−1, respectively at 25°C. The former rate constant is probably affected by substrate inhibition.

6
Since the native ferrous enzyme is capable of catalysing H abstraction from linoleic acid under aerobic but not under anaerobic conditions, while the yellow ferric enzyme is active either in the absence or presence of O2, it is proposed that O2 reversibly converts the ferrous enzyme species into a ferric state.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1977.tb11617.x

On the mechanism of action of soybean lipoxygenase-1. A stopped-flow kinetic study of the formation and conversion of yellow and purple enzyme species

The proton nuclear magnetic resonance (1H-NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of α-helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met-81 residue in glycophorin.



Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C-3 to C-11 methylene resonances. The bound dodecyl-sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate-protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1979.tb13087.x

Maarten R. Egmond

Papers

On the mechanism of action of soybean lipoxygenase-1. A stopped-flow kinetic study of the formation and conversion of yellow and purple enzyme species

1H‐Nuclear‐Magnetic‐Resonance Studies on Glycophorin and Its Carbohydrate‐Containing Tryptic Peptides

Proton NMR studies on soya bean lipoxygenase-1.