Soil Biology

Alexander P. Hansen
Devendra K. Choudhary
Pawan Kumar Agrawal
Ajit Varma Editors

Rhizobium
Biology and
Biotechnology
 Properties of Leghemoglobin (Lb)

1. Leghemoglobin (Lb) is a globular shaped protein (16 kDa, around 150

amino acids) consists of heme prosthetic group. Like blood hemoglobin,
the heme group of leghemoglobin also composed of iron porphyrin
molecule containing four modified pyrrole subunits.

2. It is red in color and found in millimolar quantities, in symbiont-

containing cells of the central tissue of legume root nodules. Therefore
middle of the nodules seems pinkish upon medial section.

3. Lb has a very high affinity for oxygen. Hence, only 48 nM

concentration of free oxygen is sufficient to cause 50% oxygenation of
Lb.

4. It was previously thought that the heme prosthetic group of plant

leghemoglobin was provided by the bacterial symbiont within symbiotic
root nodules. However, subsequent studies proved that the host plant
strongly expresses heme biosynthesis genes during the production of
globular proteins in developing nodules.

5. This globular protein usually consists of seven or eight helical

segments named A–H, with an iron group inserted between the E- and F-
helices. A space between heme and the E-helix is known as distal
pocket, is large and flexible enough to allow oxygen to coordinate with
heme iron. Oxygen combination can occur only if heme iron is in ferrous
valence state.

Reference
Chapter 15: Structure, Function, and Estimation of Leghemoglobin
Page No: 309-313
Figure: Medial section of a fresh root nodule (Top image), Porphyrin (Middle
image), 3D structure of leghemoglobin (Bottom image).
15 Structure, Function, and Estimation of Leghemoglobin 315

the respiratory chain of bacteroids, the oxygen affinity of the bacteroid oxidase
should exceed (about tenfold) the oxygen affinity of Lb. Thus, Lb is oxygenated
at the plasmalemma and carries oxygen to the symbiosome membrane. Since
there is no Lb in the peri-bacteroid membrane, oxygen has to be released at the
symbiosome membrane. This transfer of oxygen is facilitated due to the presence
of deep oxygen gradient across the peri-bacteroid membrane.

Catalase enzyme to save N2 fixation factory !!!

15.4.1 Inactivation of Lb by Oxidation

From the chemical nature of Lb, Lb3+ is expected to be continually formed in vivo
by oxidation of Lb2+ and LbO2. Soluble fraction of extracted in air shows the
presence of traces of Lb3+. A number of metabolites present in nodules, including
nitrite, superoxide radical, and peroxides, may oxidize Lb2+. Nitrite induces
oxidation of Lb2+ to Lb3+, whereas NO binds tightly to Lb2+ and LbO2 forming
nitrosyl-Lb (Lb2+.NO).
The ferrous form of Lb readily autoxidizes and is converted into ferric form
under the slightly acidic conditions of the nodules. Free radicals and other activated
oxygen species such as hydrogen peroxide react with Lb and lead to their inacti-
vation. These reactive species are generated as a result of Lb autoxidation and
mitochondrial and bacteroid respiration. Autoxidation of LbO2 varies with pH,
temperature, and concentrations of Lb; certain anions, metal anions; and chelators.
Oxidation of LbO2 to Lb3+ by a flux of superoxide radicals generated artificially and
re-reduction of Lb3+ by superoxide radicals, although at a much slower rate, were
also observed.

LbO2 ! Lb3þ þ O2  ð15:1Þ

 þ 
LbO2 þ O2 þ 2H ! Lb 3þ
þ O2 þ Lb2 O2 ð15:2Þ

Lb 3þ
þ O2 ! LbO2 ð15:3Þ

Exogenously added superoxide dismutase inhibited oxidation of LbO2 to Lb3+;

thus, it was proposed that nodule superoxide dismutase protects Lb against
inactivation.
Hydrogen peroxide attacks oxygenated Lb to generate hydroxyl radicals. When
hydrogen peroxide attacks LbO2, heme group is broken down and ferrous ion is
released, which in turn reduces hydrogen peroxide to hydroxyl free radicals. These
radicals are highly reactive and thus can oxidize nearly all types of molecules in
their vicinity, such as DNA, proteins, and unsaturated fatty acids of membranes.
There is no enzyme for scavenging these reactive species, as they are far too
reactive, but the antioxidative enzymes such as catalase can prevent their formation
by destroying hydrogen peroxide.
316 S. Singh and A. Varma

15.4.2 Restoration of the Functional State of Lb by Small

Molecules

Several researchers have suggested that a system to reduce Lb3+ should exist in
nodules. Nodules contain many potential reducing agents, such as NAD(P)H, ascor-
bate, reduced glutathione, and cysteine, to reduce Lb3+ to functional Lb. The physio-
logical levels of these reductants to determine their relative effectiveness in reducing
Lb3+ were estimated to be in the order of 150–250 μM for NADH+NADPH, 200 μM
for cysteine, 40–150 μM for reduced glutathione, and 1–2 mM for ascorbate (Becana
and Klucas 1992). Excess ascorbate, however, initially reduces Lb3+ to LbO2 but then
induces heme degradation with the formation of activated O2 species.
Flavins are intermediate electron carriers between NAD(P)H and Lb3+. In the
presence of NAD(P)H, free flavins efficiently reduce Lb3+ without formation of
superoxide or peroxide. The abundance of free flavins, especially riboflavin, plays
an important role in reducing Lb3+ in the microaerophilic conditions in nodules. NAD
(P)H supply should be relatively high and constant; otherwise with excess of flavin
and deficit of NAD(P)H, Lb2+ is oxidized back to Lb3+ (Becana and Klucas 1992).

15.4.2.1 Degradation of Lb / Process and symptom of Lb inactivation

Metabolic degradation of the Lb involves several proteases. These proteases have

high affinity for Lb. These are found in abundance in nodules entering senescence.
Degradation of Lb leads to production of two important pigments: choleglobin and
biliverdin. Choleglobin is a Lb derivative having an oxidized heme group but with
the iron still attached to it. This iron is, however, lost in biliverdin. Accumulation of
these pigments changes the color of legumes from pink to green, which acts as a
marker for Lb inactivation and therefore loss of nitrogen-fixing ability by nodules
(Becana et al. 1995).

15.5 Synthesis

Leghemoglobin synthesis starts very shortly after nodule initiation usually before
nitrogenase synthesis can be demonstrated. In legumes, the leghemoglobin synthe-
sis continues in proportion with the meristematic tissues of the nodule. In the
nodules which lack meristematic tissues (e.g., soybean, kidney bean) synthesis of
leghemoglobin occurs only for a short duration and its concentration remains
almost constant till the senescence of the nodule.
There are two known activities for ALA formation in root nodules: (1) ALA
synthase by bacterial symbiont and (2) glutamate-dependent ALA formation by the
plant host. Evidence shows that plant-derived ALA can be employed for production
of bacteroid heme. Continuous and related increase in cellular heme expression and