Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (kon > 1.4 ؋ 10 8 M ؊1 ⅐s ؊1 at 4°C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (k ‫؍‬ 2.4 ؋ 10 5 M ؊1 ⅐s ؊1 at 4°C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (k ‫؍‬ 7.1 ؎ 2 ؋ 10 8 M ؊1 ⅐s ؊1 at 4°C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission. N itric oxide (NO) typically functions in paracrine fashion; namely, NO synthesized in one cell acts on an adjacent cell to bring about a biological response such as smooth muscle relaxation involved in vasodilation (Fig. 1). NO used in signaling is synthesized by constitutive isoforms of nitric oxide synthase (NOS) that are regulated through a Ca 2ϩ ͞calmodulin interaction (1). Cell-specific external stimuli, such as bradykinin in endothelial cells and glutamate in neuronal tissue, increase cellular Ca 2ϩ levels, thereby activating a specific NOS isoform. The NO signal is amplified in an adjacent cell, where it acts on the soluble isoform of guanylate cyclase (sGC) (2, 3). Once activated, sGC converts GTP to cGMP and pyrophosphate with the subsequent second messenger action of cGMP. As illustrated in Fig. 1, increases in receptor-mediated Ca 2ϩ release in endothelial cells leads to transient activation of NOS, producing a burst of NO. NO then activates sGC in the adjacent smooth muscle, culminating in smooth muscle relaxation (vasodilation).sGC is a heterodimeric hemoprotein. The enzyme was originally isolated from bovine lung, where the subunits were designated ␣1 and ␤1. The heme moiety is the NO binding site, which, as isolated, is ferrous and 5-coordinate with a histidine residue as the protein-derived axial ligand. By using residues 1-385 of the ␤1 subunit [␤1(1-385)], the heme binding region has been localized to the ␤1-subunit with H105 as the axial heme ligand (4, 5). Steady-state analysis showed that NO binding converts the heme to a 5-coordinate nitrosyl complex in which the bond between the heme iron and the histidine has been severed. In this state, sGC is activated several hundred-fold over the basal activity. Much speculation has centered on how NO binding turns on catalytic activity. Initial ...

Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.

Phthalate dioxygenase reductase (PDR) is a prototypical iron-sulfur flavoprotein (36 kilodaltons) that utilizes flavin mononucleotide (FMN) to mediate electron transfer from the two-electron donor, reduced nicotinamide adenine nucleotide (NADH), to the one-electron acceptor, [2Fe-2S]. The crystal structure of oxidized PDR from Pseudomonas cepacia has been analyzed at 2.0 angstrom resolution resolution; reduced PDR and pyridine nucleotide complexes have been analyzed at 2.7 angstrom resolution. NADH, FMN, and the [2Fe-2S] cluster, bound to distinct domains, are brought together near a central cleft in the molecule, with only 4.9 angstroms separating the flavin 8-methyl and a cysteine sulfur ligated to iron. The domains that bind FMN and [2Fe-2S] are packed so that the flavin ring and the plane of the [2Fe-2S] core are approximately perpendicular. The [2Fe-2S] group is bound by four cysteines in a site resembling that in plant ferredoxins, but its redox potential (-174 millivolts at pH 7.0) is much higher than the potentials of plant ferredoxins. Structural and sequence similarities assign PDR to a distinct family of flavoprotein reductases, all related to ferredoxin NADP(+)-reductase.

Para-hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para-hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.

Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.

No abstract

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier Mössbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.