Ryu Makino

Role of Arg112 of Cytochrome P450cam in the Electron Transfer from Reduced Putidaredoxin ANALYSES WITH SITE-DIRECTED MUTANTS

The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0.16 s−1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were −138, −162, −182, −200, and −195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.

Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin

Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid‐point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen‐bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Pyridine and imidazole reversibly inhibit the respiratory burst in porcine and human neutrophils: evidence for the involvement of cytochrome b558 in the reaction.

Heterocyclic nitrogenous bases, such as pyridine and imidazole which bind to heme-iron in cytochromes, inhibited the respiratory burst in intact neutrophils and NADPH-dependent oxygen consumption in lysed cells. Inhibition was accompanied by a spectral change in reduced cytochrome b558 as judged by low-temperature spectroscopy at 77 K. The position and shape of the alpha-band of the cytochrome were significantly altered upon exposure to pyridine or some other bases. Both inhibition and spectral changes were reversible. The results are consistent with the view that cytochrome b558 is involved in the NADPH oxidase system in neutrophils.

Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of α- and β-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for α,β-methylene guanosine 5′-triphosphate (GMP-CPP). In contrast, 2′-dADP occupied both sites with equivalent affinities. Adenosine-5′-β,γ-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2′-dADP, probably by competing for the same site as 2′-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2′-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.

Observation of the FeIV=O stretching Raman band for a thiolate-ligated heme protein : compound I of chloroperoxidase

The FeIV=O stretching vibration has never been identified for a cysteine‐coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed‐flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm−1, which shifted to 756 cm−1 with the 18O derivative, but the ν4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin π cation radical. The second intermediate gave an intense ν4 band at 1,372 cm−1 but no oxygen isotope‐sensitive Raman band, suggesting oxygen exchange with bulk water.

Observation of the O-O stretching Raman band for cytochrome P-450cam under catalytic conditions.

Dioxygen stretching (voo) Raman band was observed for the oxy form of Pseudomonas putida cytochrome P-450 (P-450cam) generated at room temperature under catalytic conditions, that is, in the presence of D-camphor, beta-NADH, putidaredoxin, and putidaredoxin reductase, by using the mixed flow transient Raman apparatus. At the same time the visible absorption spectra were monitored for the transient species. It was found that the voo frequency is little altered by binding of putidaredoxin to P-450cam, although the reduction rate of the oxy form becomes faster. Another intermediate with an oxygen isotope-sensitive band was not found in a time region until 2 s after mixing of the reduced enzyme with oxygen.

Oxygen Binding and Redox Properties of the Heme in Soluble Guanylate Cyclase: IMPLICATIONS FOR THE MECHANISM OF LIGAND DISCRIMINATION*

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O2, thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O2 has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe2+-O2) when frozen at 77 K in the presence of O2. The ligation of O2 was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at −7 °C, with the extremely low affinity for O2. The low O2 affinity was not caused by a distal steric protein effect and by rupture of the Fe2+-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe2+-proximal His bond. Based on our results, we propose that the weak Fe2+-proximal His bond is a key determinant for the low O2 affinity of the heme moiety of soluble guanylate cyclase.

Saturated and trans-unsaturated fatty acids elicit high levels of superoxide generation in intact and cell-free preparations of neutrophils.

Saturated and trans-unsaturated fatty acids, such as laurate and elaidate, elicited O2- generation in intact porcine and human neutrophils and also in a cell-free preparation of porcine neutrophils. The activities thus induced were comparable to those induced by cis-unsaturated fatty acids. However, the activation by saturated or trans-unsaturated fatty acids was depressed almost completely in the presence of Ca2+ at around 1 mM, which is usually contained in the media for phagocytes. In contrast, the activation by cis-unsaturated fatty acids such as arachidonate was scarcely affected by Ca2+. These findings appear to demand reevaluation of the effects of long chain fatty acids on the respiratory burst system in phagocytes.

Thermodynamic aspects of the CO-binding reaction to cytochrome P-450cam. Relevance with their biological significance and structure

The CO-binding kinetics of cytochrome P-450cam(+) and P-450cam(-) have been measured in the millisecond time domain using a flash photolysis method. We have determined the reaction coordinates for free energy, enthalpy and entropy from the temperature dependence of the overall rate constants of the bimolecular forward (on) and backward (off) reactions. Comparing the thermodynamic profiles of P-450cam with that of myoglobin (Mb) reported so far, the enthalpy and the entropy coordinates exhibit the following remarkable characteristics. The CO-binding equilibrium: The stability of the CO-complex is perfectly entropy-driven for P-450cam, while enthalpy-driven for Mb. This entropy-driven feature for P-450cam is enhanced by the dissociating d-camphor. The on and off activation processes: The on and off reactions for P-450cam are dominantly controlled by the enthalpy and entropy terms, respectively, while those for Mb are rather the reverse of the case of P-450cam. The dissociation of d-camphor has a significant effect on the on reaction but no effect on the off reaction. Analyzing these thermodynamic features on the basis of the physical chemistry in the solution reaction, it was found that these characteristic profiles arise from the difference in the global structural change between the proteins. Namely, during the equilibrium process of the CO binding, this structural change is accompanied by a larger increase in the degree of freedom in P-450cam than in Mb. We discussed the correlations between the structural changes and their biological significance.

Isolation of glucosaminyl-β(1–4)-muramic acid and phosphoric acid ester of this disacoharide from acid hydrolysates of peptidoglycan of Bacillus cereus ahu 1356 cell walls

Abstract Glucosaminyl-β(1–4)-muramic acid and glucosaminyl-β(1–4)-muramic acid-6-phosphate were isolated from acid hydrolysates of Bacillus cereus AHU 1356 cell wall peptidoglycan. The yield of the disaccharide suggests that the majority of the amino groups of the glucosamine residues in this peptidoglycan are nonsubstituted.