Biomedical subjects

L A Andersson

Publications and source records attributed to L A Andersson.

23 records · Page 2Linked to original sources

PubMed1982

Endogenous cysteine ligation in ferric and ferrous cytochrome P-450. Direct evidence from x-ray absorption spectroscopy.

Extended x-ray absorption fine structure spectroscopy has been applied to the elucidation of the structure of the heme iron site of bacterial cytochrome P-450. The low spin ferric, high spin ferric, ferrous, and ferrous carbonyl states of the enzyme have been examined. Curve-fitting analysis of the data provides direct and compelling evidence for the presence of a sulfur atom in the first coordination sphere of the iron. The iron-nitrogen (porphyrin) distances indicate five coordination in high spin ferric and ferrous P-450 and six coordination in low spin ferric and ferrous carbonyl P-450. The iron-sulfur distances are consistent with thiolate ligation, presumably from cysteinate, in all four states of the enzyme. In each case, the iron-sulfur bond distance is equal to or shorter than the analogous Fe-S bonds in model iron porphyrin thiolate complexes whose crystal structures have been determined. Since known thiol-sulfur:iron-heme bond distances are noticeably longer than the corresponding thiolate bonds, the X-ray absorption fine structure results strongly suggest that, in each P-450 state examined, the sulfur donor is a thiolate. The results reported in this paper concerning the ligand identity, state of protonation, and metal-ligand bond distances are of critical importance to a complete description of the P-450 reaction cycle and its mechanism of oxygen activation.

Cysteine↗

PubMed1982

Sulfur donor ligand binding to ferric cytochrome P-450-CAM and myoglobin. Ultraviolet-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic investigation of the complexes.

The binding of thiol, thiolate, thioether, and disulfide sulfur donor ligands to ferric cytochrome P-450-CAM and myoglobin has been investigated by UV-visible absorption, magnetic circular dichroism (MCD), and EPR spectroscopy. For ferric P-450, the binding of all sulfur donors is competitive with substrate binding. Addition of thiols to P-450 leads to interconvertible thiol or thiolate-bound species depending on the thiol acidity (pKa) and the solution ph; ligation of thiols lowers their pKa by about 4 units. In contrast, only the thiolate-bound form is seen for myoglobin regardless of thiol acidity or solution pH (5.5-11.0), indicating that the heme iron of myoglobin is less electron-rich than that of P-450. Thiolate ligands show much higher affinity (Kd approximately 10(-6) M) for ferric P-450 than do thiols (Kd approximately 10(-3) M). The affinity of thioethers for P-450 (Kd approximately 10(-3) M) is pH-independent (pH 5.5-9.0). The observed disulfide coordination to P-450 represents the first example of disulfide ligation to heme iron; no significant evidence for thioether or disulfide binding to myoglobin is seen. Except for the thiolate complexes, the UV-visible and MCD spectral properties of the other sulfur donor . P-450 complexes are similar to, although distinguishable from, those of native P-450. The ferric P-450 . thiolate complexes exhibit MCD spectra resembling that of ferrous P-450 . CO; both also exhibit unique hyperporphyrin (split Soret) UV-visible spectra. The EPR spectra of all P-450 complexes examined display very narrow spread g-values such as are characteristic of native P-450, indicating that the endogenous cysteinate axial ligand is retained upon complex formation. The dissimilarities observed between P-450 and myoglobin in their reactivity toward sulfur donor ligands at least partly reflect the variation in heme iron electron density resulting from their different endogenous axial ligands and may, in turn, help to explain their respective physiological functions of oxygen activation and reversible oxygen binding.

Animals↗

PubMed1982

Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme.

An extensive series of ligand complexes of ferric cytochrome P-450-CAM has been examined by UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy in an attempt to identify the ligand trans to cysteinate in the six-coordinate resting state of the enzyme. Thus, the ligands used have been chosen to serve as models for coordination by potential endogenous amino acids and include alcohol, amide and carboxylate oxygen donors, amine, imidazole and indole nitrogen donors and disulfide, thioether, thiol, and thiolate sulfur donors. As this investigation has been by nature an empirical one, the conclusions are strengthened by the concurrent use of three different spectroscopic techniques. All of the complexes formed except those resulting from thiolate addition display spectroscopic properties that are broadly similar to those of low spin, six-coordinate P-450. Of the sulfur donor adducts, disulfide and thioether-bound P-450 have properties that are different enough in detail to distinguish them from native P-450. While the spectral features of the thiol-bound species and of low spin ferric P-450 are alike, the former are pH dependent due to interconversion to bound thiolate, whereas the latter display essentially no spectral changes with pH. Of the oxygen donor complexes, all but carboxylate have spectra that very closely match those of the resting enzyme. Adducts formed with most nitrogenous ligands, including several imidazole derivatives, exhibit spectra that are sufficiently different from native P-450 to exclude them as candidates for the sixth ligand. Interestingly, the spectral properties of a complex formed with an imidazole derivative having a bulky electron-withdrawing substituent in the alpha position are comparable to those native P-450 except for the line shape of the EPR spectrum. Previously published theoretical work suggests that the spectral differences seen between this imidazole derivative and the other examined are electronic and not steric in origin. As no similar electronic mechanism exists for the protein to reduce the electron-donating ability or histidine, it is felt that coordination of histidine in the sixth position of P-450 can be ruled out. In conclusion, close examination of all spectral data reveals that amino acid analog adducts of P-450-CAM with amides and, in particular, alcohols, produce spectra that almost exactly duplicate those of native P-450 and suggests that the ligand trans to cysteinate in the six-coordinate ferric enzyme has an oxygen donor atom.

Amino Acids↗

PubMed1980

Effects of platinum complexes on chymotrypsin.

A variety of platinum complexes were evaluated as inhibitors of alpha-chymotrypsin. A standard enzyme assay was used with benzoyl-L-tyrosine ethyl ester as the substrate. Rb2PtBr4, trans-Pt(NH3)2Cl2, and K2PtCl4 were all effective enzyme inhibitors. Pt(Gly-L-Met(Cl2, Pt(Met)(NH3)2Cl, cis-Pt(NH3)2Cl2, and two ethylenediamminedichloroplatinum complexes were weak inhibitors of alpha-chymotrypsin. A surprising finding was the noninhibition of an immobilized preparation of alpha-chymotrypsin by the above inhibitors and by active-site-directed modifying reagents.

Animals↗

PubMedDate not supplied

The influence of oxygen donor ligation on the spectroscopic properties of ferric cytochrome P-450: ester, ether and ketone co-ordination to the haem iron.

Homogeneous low-spin complexes of cytochrome P-450-CAM with esters, ethers and ketones have been prepared and characterized by u.v.-visible absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopy. Direct haem iron ligation has been verified by competition for binding with well-known haem ligands. The u.v.-visible spectra of the oxygen-donor complexes exhibit near-u.v. (delta) transitions near 356 nm, Soret maxima at 417 +/- 1 nm, beta bands near 536 nm and alpha peaks near 569 nm, with alpha greater than beta in intensity. Negative delta and Soret CD troughs are seen. The MCD spectra have minima at approximately 356 nm, intense derivative-shaped Soret features centred at approximately 416 nm and four characteristic features beyond 450 nm. The EPR spectra of these complexes, while similar to that of the native enzyme, exhibit slight variances. Anomalous spectral and substrate binding properties have been reported in the study of cytochrome P-450 under conditions employing solvents and non-phosphate buffers containing oxygen functionalities, and have been attributed to 'solvent effects'. The present work, in combination with our previous report of alcohol, amide and carboxylate oxygen donor complexes of cytochrome P-450, is evidence that a wide variety of oxygen-donor species are capable of direct ligation to the haem iron of cytochrome P-450. This leads us to suggest oxygen-donor ligation to cytochrome P-450 as the origin of spectral and substrate binding anomalies previously attributed to solvent effects.

Circular Dichroism↗