Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands.
We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity. (+info)
EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase.
The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding. (+info)
Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1.
The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry. (+info)
Altered ligand rebinding kinetics due to distal-side effects in hemoglobin chico (Lysbeta66(E10) --> thr).
Hb Chico is an unusual human hemoglobin variant that has lowered oxygen affinity, but unaltered cooperativity and anion sensitivity. Previous studies showed these features to be associated with distal-side heme pocket alterations that confer increased structural rigidity on the molecule and that increase water content in the beta-chain heme pocket. We report here that the extent of nanosecond geminate rebinding of oxygen to the variant and its isolated beta-chains is appreciably decreased. Structural alterations in this variant decrease its oxygen recombination rates without significantly altering rates of migration out of the heme pocket. Data analysis indicates that one or more barriers that impede rebinding of oxygen from docking sites in the heme pocket are increased, with less consequence for CO rebinding. Resonance Raman spectra show no significant alterations in spectral regions sensitive to interactions between the heme iron and the proximal histidine residue, confirming that the functional differences in the variant are due to distal-side heme pocket alterations. These effects are discussed in the context of a schematic representation of heme pocket wells and barriers that could aid the design of novel hemoglobins with altered ligand affinity without loss of the normal allosteric responses that facilitate unloading of oxygen to respiring tissues. (+info)
Absence in amphotericin B-spiked human plasma of the free monomeric drug, as detected by SERS.
Using surface enhanced Raman spectroscopy (SERS) which enables us to specifically detect traces of monomeric amphotericin B (AmB), we were able to show that in a 10(-5) M AmB suspension, the concentration of free drug was below 10(-8) M in the presence of low density lipoproteins (LDL) or plasma. The affinity constant of AmB for LDL determined from electronic absorption data, was found to be 4 x 10(6) M(-1). Therefore, since AmB appears to be in the majority bound to lipoproteins under in vivo conditions, its toxicity should not result from the induction of host-cell transmembrane permeability but rather from the internalization of the AmB-LDL complex. (+info)
Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.
The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes. (+info)
An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.
Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5. (+info)
Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2'-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2'-deoxycytidine.
UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2'-deoxycytidine (HO5dCyt) in its anionic form. In DNA, this 2'-deoxycytidine analog arises from the oxidation of 2'-deoxycytidine and induces C --> T transitions with 10(2) greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (approximately 1650 cm-1) is enhanced at approximately 65 degrees C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N3-methylcytidine at high pH and N4-methoxy-2'-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO5dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations. (+info)