Steve L. Alam

Detailed NMR Analysis of the Heme-Protein Interactions in Component IV Glycera dibranchiata Monomeric Hemoglobin-CO

Complete 13C, 15N, and 1H resonance assignments have been obtained for the recombinant, ferrous CO-ligated form of component IV monomeric hemoglobin from Glycera dibranchiata. This 15642 Da myoglobin-like protein contains a large number of glycine and alanine residues (47) and a heme prosthetic group. Coupling constant information has allowed the determination of χ1 and χ2 torsion angles, backbone φ angles, as well as 43 of 81 possible assignments to Hβ2/β3 pairs. The 13Cα, 13Cβ, 13C′, and 1Hα assignments yield a consensus chemical shift index (CSI) that, in combination with NOE information and backbone torsion angles, defines seven distinct helical regions for the proteins global architecture. Discrepancies between the CSI and NOE/3JHNHα-based secondary structure definitions have been attributed to heme ring current shifts on the basis of calculations from a model structure [Alam et al. (1994) J. Protein Chem., 13, 151-164]. The agreement can be improved by correcting the 1Hα chemical shifts for the ring current contributions. Because the holoprotein was assembled from isotopically enriched globin and natural isotope-abundance heme, data from 13C-filtered/13C-edited and 13C-filtered/13C-filtered 2D NOESY experiments could be used to determine complete heme proton assignments and to position the heme within the protein. The results confirm the unusual presence of Phe31(B10) and Leu58(E7) side chains near the heme ligand binding site which may alter the polarity and steric environment and thus the functional properties of this protein.

Proton NMR Studies of Selected Paramagnetic Heme Proteins

Among the heme proteins three types have attracted our attention during the past thirteen years. Not all of the heme proteins currently studied in this laboratory are paramagnetic in their native states, but several are. The presence of the heme-containing, redox-active iron ion makes it possible to study paramagnetic forms of all of these proteins. NMR data for three of these proteins will be the topic of this article, which is written from the point of view of relating experiences encountered in this laboratory. One of the first issues faced in reading the NMR literature is realizing that two heme numbering systems are simultaneously in use. The modified Fischer scheme seems to be preferred in the U.S. literature and is also widely employed by porphyrin chemists. The IUB/IUPAC scheme is more widely used outside the U.S. Examples of both are presented in Fig. 1.

Recombinant Perdeuterated Protein as an Efficient Method for Making Unambiguous Heme Proton Resonance Assignments: Cyanide-Ligated Glycera Dibranchiata Monomer Methemoglobin Component IV as an Example

In order to overcome the difficulties of selectively assigning the heme proton resonances in paramagnetic low-spin heme proteins, a method involving perdeuteration of the globin has been developed. This method allows rapid proton assignments of the heme prosthetic group to be made, however its use is restricted to proteins for which a suitable expression system exists. As an example of this method, the process of making complete heme proton assignments of the cyanide-ligated Glycera dibranchiata monomer hemoglobin Component IV in both the naturally protonated, native protein and the recombinant, perdeuterated protein is presented. There are many potential uses of this method aside from heme-containing proteins.

Proton Hyperfine Resonance Assignments in Glycera Dibranchiata Monomer Hemoglobin Component IV

Publisher Summary This chapter discusses the proton hyperfine resonance assignments in Glycera dibranchiata monomer hemoglobin component IV. The low-spin, cyanide-ligated Fe 3+ form of monomer Component IV are chosen for initial studies. In this paramagnetic form, the heme iron ion acts as an intrinsic shift reagent. The result is a proton nuclear magnetic resonance (NMR) spectrum that in D 2 O is spread over ∼27 ppm at 30°C, with the most highly shifted resonances due to protons located closest to the heme iron ion. In this way, a selective view of the heme site can be obtained. The hyperfine shifted resonances are extremely sensitive to structural changes in the heme pocket, and when they are completely assigned, they are expected to be effective spectroscopic probes of heme pocket integrity in expressed proteins. The chapter presents the heme coordination structure in stereo, including the orientations of the heme/proximal histidine plane for GMH4 in comparison to sperm whale myoglobin. Being monomelic hemoglobin, GMH4 is frequently compared with myoglobin because these two proteins share very similar architectures. An important aspect of the GMH4 structure is that primary sequence results and a model built from that sequence provide evidence for an unusual heme pocket.

Proton Nuclear Magnetic Resonance Studies of Non-Covalent Complexes of Yeast Cytochrome c Peroxidase with Cytochromes c

Publisher Summary This chapter discusses proton nuclear magnetic resonance (NMR) studies of noncovalent complexes of yeast cytochrome c peroxidase with cytochromes. Cytochrome c peroxidase (CcP) from Bakers yeast catalyzes the hydrogen peroxide oxidation of ferrous cytochrome c. The enzyme mechanism includes intermediate steps involving a single electron transfer from ferrous cytochrome c to each oxidized CcP intermediate. Such electron transfers are each characterized by the net oxidation of ferrous cytochrome c, producing ferricytochrome c. A key development in understanding CcP behavior was the formulation of a CcP:cytochrome c complex model by Poulos and Kraut; this model has stimulated a great deal of work on CcP complexes. NMR has proven uniquely capable for detecting and characterizing CcP:cytochrome c complexes. Many other spectroscopic techniques have been employed in studies of these types of complexes but have provided less specific information. The degree of sophistication in exploiting modern NMR methods for studying these complexes is now in a period of growth. The chapter describes the different kinds of CcP:cytochrome c complexes currently being investigated in laboratory. It also describes the scope and limitations of studying these complexes by proton NMR spectroscopy.