Recent developments in the 13 C NMR spectroscopic analysis of paramagnetic hemes and heme proteins

Abstract

Despite the wealth of information that has been obtained from the study of paramagnetic hemes and heme proteins by 1H NMR spectroscopy, there are certain limitations imposed by the nature of paramagnetically affected resonances that are difficult to overcome. Although it has long been recognized that 13C NMR spectroscopy is likely to be a powerful complementary technique to overcome some of these limitations, the low sensitivity and low natural abundance of 13C nuclei has resulted in a lag in the application of 13C NMR spectroscopy to the study of paramagnetic hemes and heme proteins. The tremendous advances in methodology and instrumentation witnessed in the NMR field, coupled to the advent of recombinant DNA methods that have made possible the preparation and purification of significant quantities of proteins, and the biosynthesis of 13C-labeled heme, have contributed to an increased interest in the study of paramagnetic heme active sites by 13C NMR spectroscopy. As a consequence, 13C NMR spectroscopy is emerging as a powerful tool to study heme electronic structure and structure-function relationships in heme-containing proteins. In this report we strive to summarize some of the recent developments in the analysis of paramagnetic hemes and heme-containing proteins by 13C NMR spectroscopy.

The Heme Biosynthesis Pathway Is Essential for Plasmodium falciparum Development in Mosquito Stage but Not in Blood Stages

“…1). The metabolic condensation of eight 5-[ 13 C 4 ]ALA molecules to form the porphyrin macrocycle results in a stable 8-Da increase in the molecular mass of heme, PPIX, and CPP relative to unlabeled masses for these analytes, because no carbon atoms derived from the C 4 carbon of ALA are lost from decarboxylations en route to heme (36). This mass increase can be used to specifically detect and distinguish newly synthesized heme from the huge background of host-derived unlabeled heme.…”

The Heme Oxygenase(s)-Phytochrome System of Pseudomonas aeruginosa

“…strategies outlined in previous work (9,21,22). The 1 H resonance assignments corresponding to heme substituents were subsequently obtained from the NOESY map shown in Fig.…”

Isotopic labeling of the heme cofactor in cytochrome p450 and other heme proteins

“…Additionally, certain P450 enzymes (and other heme proteins) have covalently bound heme cofactors (LeBrun et al 2002), which precludes the use of techniques involving reconstitution. Woodward et al (2007) developed an E. coli system in which modified hemes may be incorporated through media supplementation, however aminolevulinic acid cannot be used as a source of heme precursor, as the inactive gene product in this strain is downstream from ALA synthesis in the biosynthetic pathway (Rivera and Caignan 2004). In addition, cultures must be grown anaerobically, which necessitates the use of more sophisticated culture facilities.…”