Biomedical subjects
Publications and source records attributed to Michael A Noble.
Two Canadian urban areas received travelers with severe acute respiratory syndrome (SARS) before the World Health Organization issued its alert. By July 2003, Vancouver had identified 5 cases (4 imported); Toronto reported 247 cases (3 imported) and 43 deaths. Baseline preparedness for pandemic threats may account for the absence of sustained transmission and fewer cases of SARS in Vancouver.
The interaction of nitric oxide with cytochrome P450 BM3 from Bacillus megaterium has been analyzed by spectroscopic techniques and enzyme assays. Nitric oxide ligates tightly to the ferric heme iron, inducing large changes in each of the main visible bands of the heme and inhibiting the fatty acid hydroxylase function of the protein. However, the ferrous adduct is unstable under aerobic conditions, and activity recovers rapidly after addition of NADPH to the flavocytochrome due to reduction of the heme via the reductase domain and displacement of the ligand. The visible spectral properties revert to that of the oxidized resting form. Aerobic reduction of the nitrosyl complex of the BM3 holoenzyme or heme domain by sodium dithionite also displaces the ligand. A single electron reduction destabilizes the ferric-nitrosyl complex such that nitric oxide is released directly, as shown by the trapping of released nitric oxide. Aerobically and in the absence of exogenous reductant, nitric oxide dissociates completely from the P450 over periods of several minutes. However, recovery of the nativelike visible spectrum is accompanied by alterations in the catalytic activity of the enzyme and changes in the resonance Raman spectrum. Specifically, resonance Raman spectroscopy identifies the presence of internally located nitrated tyrosine residue(s) following treatment with nitric oxide. Analysis of a Y51F mutant indicates that this is the major nitration target under these conditions. While wild-type P450 BM3 does not form an aerobically stable ferrous-nitrosyl complex, a site-directed mutant of P450 BM3 (F393H) does form an isolatable ferrous-nitrosyl complex, providing strong evidence for the role of this residue in controlling the electronic properties of the heme iron. We report here the spectroscopic characterization of the ferric- and ferrous-nitrosyl complexes of P450 BM3 and describe the use of resonance Raman spectroscopy to identify nitrated tyrosine residue(s) in the enzyme. Nitration of tyrosine in P450 BM3 may exemplify a typical mechanism by which the ubiquitous messenger molecule nitric oxide exerts a regulatory function over the cytochromes P450.
The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.
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The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).
P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties.