Bovine carbonyl lactoperoxidase structure at 2.0Å resolution and infrared spectra as a function of pH.

Lactoperoxidase (LPO) is a hemeprotein catalyzing the oxidation of thiocyanate and I(-) into antimicrobials and small aromatic organics after being itself oxidized by H(2)O(2). LPO is excreted by the lungs, mammary glands, found in saliva and tears and protects mammals against bacterial, fungal and viral invasion. The Fe(II) form binds CO which inactivates LPO like many other hemeproteins. We present the 3-dimensional structure of CO-LPO at 2.0Å resolution and infrared (IR) spectra of the iron-bound CO stretch from pH 3 to 8.8 at 1 cm(-1) resolution. The observed Fe-C-O bond angle of 132° is more acute than the electronically related Fe(III), CN-LPO with a Fe-C-N angle of 161°. The orientations of the two ligands are different with the oxygen of CO pointing towards the imidazole of distal His109 while the nitrogen of CN points away, the Fe(II) moves towards His109 while the Fe(III) moves away; both movements are consistent with a hydrogen bond between the distal His109 and CO, but not to the nitrogen of CN-LPO. The IR spectra of CO-LPO exhibit two major CO absorbances with pH dependent relative intensities. Both crystallographic and IR data suggest proton donation to the CO oxygen by His109 with a pK ≈ 4; close to the pH of greatest enzyme turnover. The IR absorbance maxima are consistent with a first order correlation between frequency and Fe(III)/Fe(II) reduction potential at pH 7; both band widths at half-height correlate with electron density donation from Fe(II) to CO as gauged by the reduction potential.

Enzymatic cascade reactions inside polymeric nanocontainers: a means to combat oxidative stress.

Oxidative stress, which is primarily due to an imbalance in reactive oxygen species, such as superoxide radicals, peroxynitrite, or hydrogen peroxide, represents a significant initiator in pathological conditions that range from arthritis to cancer. Herein we introduce the concept of enzymatic cascade reactions inside polymeric nanocontainers as an effective means to detect and combat superoxide radicals. By simultaneously encapsulating a set of enzymes that act in tandem inside the cavities of polymeric nanovesicles and by reconstituting channel proteins in their membranes, an efficient catalytic system was formed, as demonstrated by fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. Superoxide dismutase and lactoperoxidase were selected as a model to highlight the combination of enzymes. These were shown to participate in sequential reactions in situ in the nanovesicle cavity, transforming superoxide radicals to molecular oxygen and water and, therefore, mimicking their natural behavior. A channel protein, outer membrane protein F, facilitated the diffusion of lactoperoxidase substrate/products and dramatically increased the penetration of superoxide radicals through the polymer membrane, as established by activity assays. The system remained active after uptake by THP-1 cells, thus behaving as an artificial organelle and exemplifying an effective approach to enzyme therapy.