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From Proteopedia

Revision as of 01:03, 6 November 2012

Leghemoglobin

Introduction

Leghemoglobin is a protein that is required by Legume plants in order to fix nitrogen by participating in the Nitrogen Fixation pathway. It is found in legumes such as cowpea, soybeans, alfalfa, and other different types of beans. Leghemoglobin functions by keeping the oxygen at a specific low concentration to the bacteroids to allow for respiration, but at the same time, this low concentration keeps the oxygen out of the nitrogen cycle so it doesn’t inhibit the nitrogenase activity (Ref 1).

Leghemoglobin is a 16 kD globin protein containing eight secondary alpha helices, constituted by a heme group(protoporphyrin XI) and a single polypeptide. In its ferrous (reduced state), Leghemoglobin binds oxygen for transportation. (Ref 5). It is hypothesized that Leghemoglobin can be formed by one of the two ways:

1. The Rhizobium bacteroid (Ref 2) is thought to synthesize the heme group in the root nodules of the legume, where it then gives the plant the heme to complete the synthesizing of the Lb (Ref 1).
2. The plant itself has also been thought to possibly produce the heme itself, in the mytochondria of the plant cells. It then combines with the peptide sequence to complete the whole protein (Ref 1).

Function

Structure

The 16 kDa polypeptide leghemoglobin consists of two main subunits, the main globin structure and the iron-encompassing heme (protoporphyrin XI) group. These two subunits form a molecule structurally similar to Myoglobin (Ref 1). The globin fold portion of the molecule is the standard globin secondary structure, a series of 8 alpha helices (Ref 5). Depending on the species and specific type of Leghemoglobin, the primary amino acid sequence can differ some, but overall it is well conserved.

The heme prosthetic group has been found to be largely the same in different proteins and in different species (Ref 1). The main difference is a considerably larger heme group in Leghemoglobin than its other oxygen-transferring globin counterparts. The way the heme group attaches to the polypeptide is also different from myoglobin and hemoglobin. The steric crowding around the ligand binding site (beside the heme) is less than the other proteins, plus the distal and proximal histidine residues are in different orientations (Ref 7). This makes the oxygen affinity larger than that of Myoglobin and Hemoglobin (Ref 8). This heme group, more specifically, consists of four 5-membered pyrrole rings, forming a cyclic ring around a central iron (Fe) atom. This atom is contained within four equatorial nitrogens, in addition to another nitrogen from a close histidine residue and an opposite dioxygen (Ref 9). The ligand contact residues are Phe30, His63, His97, and Val67 (Ref 7). ***Add a section about H-bonding of the ligand with the Lb and how the heme group fits into the molecule***

The methods by which various Leghemoglobins were purified, and then analyzed, are as follows:

• Ammonium Sulfate Precipitation
• Concentration by Ultrafiltration
• Electrophoresis
• Anion-exchange Column Chromatography
• Isoelectrofocusing
• Nuclear Magnetic Radiance (NMR)
• X-ray crystallography (Ref 1 and 6)