Aspects of the hemes and modulation of hydrogen donors in catalases from bovine liver, yeast, and escherichia coli

Catalase is the enzyme which decomposes hydrogen peroxide to water and oxygen. Escherichia coli contains two catalases. Hydroperoxidase I (HPI) is a bifunctional catalase-peroxidase. Hydroperoxidase II (HPII) is only catalytically active toward H202. Expression of the genes encoding these proteins is controlled by different regimes. HPJI is thought to be a hexamer, having one heme d cis group per enzymatic subunit. HPII wild type protein and heme containing mutant proteins were obtained from the laboratory of P. Loewen (Univ. of Manitoba). Mutants constructed by oligonucleotidedirected mutagenesis were targeted for replacement of either the His128 residue or the Asn201 residue in the vicinity of the HPII heme crevice. His128 is the residue thought to be analogous to the His74 distal axial ligand of the heme in the bovine liver enzyme, and Asn201 is believed to be a residue critical to the function of the enzyme because of its role in orienting and interacting with the substrate molecule. Investigation of the nature of the hemes via absorption spectroscopy of the unmodified catalase proteins and their derived pyridine hemochromes showed that while the bovine and Saccharomyces cerevisiae catalase enzymes are protoheme-containing, the HPII wild type protein contains heme d, and the mutant proteins contain either solely protoheme, or heme d-protoheme mixtures. Cyanide binding studies supported this, as ligand binding was monophasic for the bovine, Saccharomyces cerevisiae, and wild type HPII enzymes, but biphasic for several of the HPII mutant proteins. Several mammalian catalases, and at least two prokaryotic catalases, are known to be NADPH binding. The function of this cofactor appears to be the prevention of inactivation of the enzyme, which occurs via formation of the inactive secondary catalase peroxide compound (compound II). No physiologically plausible scheme has yet been proposed for the NADPH mediation of catalase activity. This study has shown, via fluorescence and affinity chromatography techniques, that NADPH binds to the T (Typical) and A (Atypical) catalases of Saccharomyces cerevisiae, and that wild type HPII apparently does not bind NADPH. This study has also shown that NADPH is unlike any other hydrogen donor to catalase, and addresses its features as a unique donor by proposing a mechanism whereby NADPH is oxidized and catalase is protected from inactivation via the formation of protein radical species. Migration of this radical to a position close to the NADPH is also proposed as an adjunct hypothesis, based on similar electron migrations that are known to occur within metmyoglobin and cytochrome c peroxidase when reacted with H202. Validation of these hypotheses may be obtained in appropriate future experiments.

Pigment orientation changes detected by low temperature linear dichroism spectroscopy: cold-hardening transition in phycobilisome-containing organisms

Low temperature (77K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. pee 6301, and cold-hardening in winter rye (Secale cereale L. cv. Puma). Samples were oriented for spectroscopy using the gel squeezing method (Abdourakhmanov et aI., 1979) and brought to 77K in liquid nitrogen. The linear dichroism (LD) spectra of Synechococcus 6301 phycobilisome/thylakoid membrane fragments cross-linked in light state 1 and light state 2 with glutaraldehyde showed differences in both chlorophyll a and phycobilin orientation. A decrease in the relative amplitude of the 681nm chlorophyll a positive LD peak was observed in membrane fragments in state 2. Reorientation of the phycobilisome (PBS) during the transition to state 2 resulted in an increase in core allophycocyanin absorption parallel to the membrane, and a decrease in rod phycocyanin parallel absorption. This result supports the "spillover" and "PBS detachment" models of the light state transition in PBS-containing organisms, but not the "mobile PBS" model. A model was proposed for PBS reorientation upon transition to state 2, consisting of a tilt in the antenna complex with respect to the membrane plane. Linear dichroism spectra of PBS/thylakoid fragments from the red alga, Porphyridium cruentum, grown in green light (containing relatively more PSI) and red light (containing relatively more PSll) were compared to identify chlorophyll a absorption bands associated with each photosystem. Spectra from red light - grown samples had a larger positive LD signal on the short wavelength side of the 686nm chlorophyll a peak than those from green light - grown fragments. These results support the identification of the difference in linear dichroism seen at 681nm in Synechococcus spectra as a reorientation of PSll chromophores. Linear dichroism spectra were taken of thylakoid membranes isolated from winter rye grown at 20°C (non-hardened) and 5°C (cold-hardened). Differences were seen in the orientation of chlorophyll b relative to chlorophyll a. An increase in parallel absorption was identified at the long-wavelength chlorophyll a absorption peak, along with a decrease in parallel absorption from chlorophyll b chromophores. The same changes in relative pigment orientation were seen in the LD of isolated hardened and non-hardened light-harvesting antenna complexes (LHCII). It was concluded that orientational differences in LHCII pigments were responsible for thylakoid LD differences. Changes in pigment orientation, along with differences observed in long-wavelength absorption and in the overall magnitude of LD in hardened and non-hardened complexes, could be explained by the higher LHCII monomer:oligomer ratio in hardened rye (Huner et ai., 1987) if differences in this ratio affect differential light scattering properties, or fluctuation of chromophore orientation in the isolated LHCII sample.