Porphyrins

Tamara Božina

Contents of this lecture

• Introduction - porphyrins structure and nomenclature

- heme structure

• Heme biosynthesis

- characteristic reactions and enzymes

- regulation of biosynthesis

- disorders in heme biosynthesis

• Heme degradation

- characteristic reactions and enzymes

- disorders in bilirubin metabolism

Hemoproteins

Introduction

Hemoprotein is a protein that contains a heme prosthetic group. They are very large class of metalloproteins and have diverse biological functions including oxygen transport, which is completed via hemeproteins including hemoglobin and myoglobin. Some hemoproteins - cytochrome P450s, cytochrome c oxidase, catalase and peroxidases are enzymes. They often activate O2 for oxidation or hydroxylation.

Hemoproteins also enable electron transfer as they form part of the electron transport chain. Cytochrome a, cytochrome b, and cytochrome c have such electron transfer functions.

Porphyrins - structure and chemical properties

• a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methene bridges (=CH―)

• metal complexes derived from porphyrins occur naturally

• the presence of carboxyl groups of the substituents makes the porphyrins soluble in water

• porphyrins typically absorb strongly in the visible region of the electromagnetic spectrum, i.e. they are deeply colored

• porphyrins have been evaluated in the context of photodynamic therapy (PDT) since they strongly absorb light, which is then converted to energy and heat in the illuminated areas (cancer treatment)

porphin – the simplest porphyrin

heme

Introduction

pyrrole

The Fischer system for nomenclature of porphyrins

• a pyrrole ring are indicated by A, B, C and D (I, II, III and IV in older literature)

• methene bridges (=CH―) are indicated by α, β, γ i δ

• positions of the substituents are numbered 1-8

Hans Fischer

P = propionyl group (–CH2CH2COOH) M = methyl group (–CH3) V = vinyl group (–CH=CH2)

Introduction

1 2

3

4

5 6

7

8

porphyrin

(C20H14N4)

protoporphyrin III (IX)

• the name of the porphyrins of interest consist of a WORD and a NUMBER, e.g., uroporphyrin III. The word denotes the kinds of substituents found on the ring, and the number denotes how they are arranged

• three important words that form the name of the porphyrin:

– uroporphyrin: substituents are A (–CH2COOH) and P (–CH2CH2COOH)

– coproporphyrin: substituents are M (–CH3) and P

– protoporphyrin: substituents are M, P and V (–CH=CH2)

• two important numbered series: series I i III (series II and IV do not occur in nature)

– series I: the substituents repeat in a regular manner, e.g. APAPAPAP

– series III: the order of substituents is reversed (the substituents on ring IV were replaced with respect to series I), npr. APAPAPPA

Introduction

The Fischer system for nomenclature of porphyrins

protoporphyrin III (IX)

parent porphyrin of heme

uroporphyrin I uroporphyrin III coprporphyrin I coproporphyrin III

Heme structure

Introduction

Heme B

Various heme types (differences in peripheral substituents):

• Heme a (present in cytochrome c oxidase, has a long isoprenoid tail attached to one of the five-membered rings)

• Heme b (present in hemoglobin and myoglobin)

• Heme c (present in cytochrome c, covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues)

Introduction

Heme b in hemoglobin - a reminder of what you heard at the seminar „Hemoglobin and myoglobin” !!!

Protoporphyrin IX

Heme structure

4 methyl groups

2 vinyl groups

2 propionyl groups

A heme group consists of an iron (Fe) ion held in a

heterocyclic ring, known as a porphyrin. This porphyrin

ring consists of four pyrrole molecules cyclically linked

together (by methine bridges) with the iron ion bound

in the center. The iron ion, which is the site of oxygen

binding, coordinates with the four nitrogen atoms in

the center of the ring, which all lie in one plane. The

iron is bound strongly (covalently) to the globular

protein via the N atoms of the imidazole ring of F8

histidine residue (also known as the proximal histidine)

below the porphyrin ring. A sixth position can reversibly

bind oxygen by a coordinate covalent bond, completing

the octahedral group of six ligands.

Heme biosynthesis

Tissue localization:

• marrowbone (85 %)

• liver

Intracellular localization:

• mitochondria

• cytosol

1. Condensation of glycine and succinyl-CoA (mitochondria)

Heme biosynthesis

2. Porphobilinogen synthesis (cytosol)

M

P

P

P

P

M

M M

M

P

P

P

P

M

M

M

Heme biosynthesis

Abbreviations of substituents A = acetate P = propionyl M = methyl V = vinyl

methylene bridges

(marked in red)

3. Uroporphobilinogen III and coproporphobilinogen III synthesis (cytosol)

protoporphyrinogen IX

4. Heme synthesis (mitochondria)

M

P

P

P

P

M

M M

M

P

P M

M M

V

V

M

P

P M

M M

V

V

2 CO2

methene bridges

Heme biosynthesis

P = propionyl M = methyl V = vinyl

heme b

coproporphyrinogen oxidase

protoporphyrinogen oxidase

protoporphyrin III (IX)

Regulation of heme biosynthesis

δ-aminolevulinate-synthase 1 (ALAS1) –

a key regulatory enzyme for heme biosynthesis in the liver

allosteric inhibitor: hem (feedback inhibition)

induction of synthesis by drugs (phenobarbital,

4-aminosalicylic acid...)

Pb – inhibitor of ALA-dehydratase and ferrochelatase

ALAS2 – erythroid-specific isoform, in erythroid cells does not represent the regulatory step of heme synthesis

Heme biosynthesis

PBG-synthase

Disorders in heme biosynthesis

PORPHYRIA

• partial or complete deficiency of an enzyme involved in the biosynthesis of various porphyrins and heme

• pathological accumulation of biosynthetic pathway intermediates

• heritable

• acquired

Symptoms:

• photosensitivity of the skin - painful blisters are formed that leave scars on the skin

• red colored urine - excretion of porphyrinogen

• brown-red fluorescence of teeth under UV light

• enhanced hair growth on the face and limbs

• neurological dysfunction (hysteria, manic depression ...)

Disorders in heme biosynthesis

According to the major site of increased accumulation of porphyrins or their precursors are divided into:

a) hepatic (AIP, PCT, HCP i VP)

b) erythropoietic (CEP, EPP)

According to their clinical features:

a) acute (AIP, HCP, VP)

b) cutaneous (CEP, PCT, EPP)

Disorders in heme biosynthesis

Summary of major findings in porphyrias

Disorders in heme biosynthesis

(spleen, liver, bone marrow)

Heme degradation

• normal human erythrocyte has a life span of about

100 - 120 days

• globin chain is degraded into amino acids

• iron is reused

• hem is translated into a soluble chain form and

excreted

• 75% of bilirubin comes from hemoglobin, while

the rest is non-hemoglobin origin (myoglobin,

cytochrome, catalase)

Heme degradation

old erytrocytes

Hemoglobin

Aminoacids

Heme

Excretion

Heme is the source of bile pigments

Heme degradation

• 1st step: conversion of heme to biliverdin (cleavage of

α-methene bridge to form a linear tetrapyrrole). The

other products of the reaction are free Fe2+ and CO.

• 2nd step: conversion of biliverdin to bilirubin (the

central methene bridge of biliverdin is reduced to

form bilirubin)

The formation of the heme-degradation products biliverdin and bilirubin is responsible for the color of bruises.

Heme

heme-oxigenase

biliverdin-reductase

Conjugation of bilirubin in the liver

Heme degradation

Bilirubin-diglucuronide

(conjugated bilirubin)

P P

UDP-glucuronyl transferase

kidney bacteria

Stercobilin Urobilin

Urobilinogen

E = ethyl P = propionyl M = methyl V = vinyl

2 UDP

2 UDP-glucuronic acid

β-glukuronidase

Bilirubin is largely insoluble, and it travels in the bloodstream as a complex with serum albumin. In the liver, bilirubin is transformed to the bile pigment bilirubin diglucuronide. This product is sufficiently water-soluble to be secreted with other components of bile into the small intestine, where microbial enzymes convert it to several products, predominantly urobilinogen. Some urobilinogen is reabsorbed into the blood and transported to the kidney, where it is converted to urobilin, the compound that gives urine its yellow color. Urobilinogen remaining in the intestine is converted (in another microbe-dependent reaction) to stercobilin, which imparts the red-brown color to feces.

Disorders in bilirubin metabolism

Disorders in bilirubin metabolism

Impaired liver function or blocked bile secretion causes bilirubin to leak from the liver into the blood, resulting in a yellowing of the skin and eyeballs, a condition called JAUNDICE.

Disorders in bilirubin metabolism

HYPERBILIRUBINEMIA – a condition in which there is an elevated level of bilirubin in the blood (> 20 μmol/L). A sufficient elevation of bilirubin leads to JAUNDICE.

HEMOLYTIC (prehepatic) – caused by an excessive red cell breakdown which overwhelms the liver’s ability to conjugate bilirubin.

acute and chronic hemolytic anemia

neonatal jaundice

HEPATOCELLULAR (hepatic) – there is dysfunction of the hepatic cells. The liver loses the ability to conjugate bilirubin.

Crigler-Najjar syndrome, Gilbert's syndrome: a rare inherited disorders of impaired

bilirubin conjugation

OBSTRUCTIVE (posthepatic) – caused by an interruption to the drainage of bile containing conjugated bilirubin in the biliary system

Dubin-Johnson syndrome, Rotor syndrome: a rare, relatively benign, autosomal recessive

bilirubin disorders, associated with a defect in the ability of hepatocytes to secrete

conjugated bilirubin into the bile

Disorders in bilirubin metabolism

Hemolytic (prehepatic) jaundice

Excessive hemolysis of erythrocytes

Increased synthesis of bilirubin

Disorders in bilirubin metabolism

Type of jaundice

Serum bilirubin Colour of

stool Urine

Total Unconjugated Urobilinogen Bilirubin

hemolitic ↑ normal dark ↑ -

Hepatocellular (hepatic) jaundice

Liver damage

Disruption of conjugation and transport of bilirubin

Disorders in bilirubin metabolism

Type of jaundice Serum bilirubin Colour of

stool Urine

Total Conjugated Urobilinogen Bilirubin

hepatocellular ↑ ↑ pale ↑ +

Obstructive (posthepatic) jaundice

Obstruction of the gallbladder

The inability of bilirubin to enter the intestines

Disorders in bilirubin metabolism

Type of jaundice

Serum bilirubin Colour of

stool Urine

Total Conjugated Urobilinogen Bilirubin

obstructive ↑↑ ↑ acholic - +

• newborn infants sometimes develop jaundice because they have not yet produced enough glucuronyl bilirubin transferase to process their bilirubin.

• occurs as a result of the breakdown of fetal hemoglobin to be replaced by adult Hb and also as a result of liver immaturity

• decreased activity of UDP-glucuronyl transferase

↑ conc. of unconjugated bilirubin are toxic to the central nervous system bilirubin toxic encephalopathy (kernicterus)

• a traditional treatment to reduce excess bilirubin, exposure to a fluorescent lamp, causes a photochemical conversion of bilirubin to compounds that are more soluble and easily excreted.

Neonatal jaundice

Disorders in bilirubin metabolism

Key terms to remember

• hemoproteins • difference porphyrinogens / porphyrins • heme biosynthesis - tissue and intracellular localization • δ-aminolevulinate-synthase 1 - a key regulatory enzyme for heme biosynthesis in the liver • porphyria • biliverdin • difference unconjugated bilirubin / conjugated bilirubin • urobilinogen • stercobilin, urobilin • hyperbilirubinemia / jaundice

Literature • Victor W. Rodwell, David A. Bender, Kathleen M. Botham, Peter J. Kennelly, P. Harper's

Illustrated Biochemistry, 28th ed. 2011. • D.L. Nelson i M.M. Cox: Lehninger Principles of Biochemistry, 4th ed, Worth Publishers,

USA, 2005.