edited bv: MICHAELR. SLABAUGH Weber State College Ogden. Utah 84408
chem I ~upplement
The Biochemistry of Some Iron Porphyrin Complexes Colin J. Rlx Royal Melbourne Institute of Technology, 124 LaTrobe Street, Melbourne, Vic. 3000. Australia
Why do Traffic police on point duty in New York and Tokyo take oxygen rests? ( I ) Babies born to mothers who are heavy smokers weigh about 250 g (5%) lighter than average? (2) Poorly stored beet type vegetables cause sickness and sometimes death in babies and children who consume them? (3) Butchers "pumpWmeatwith brine containing sodium nitrite? ( 4 ) Many people die from smoke inhalation rather than the crash impact during an aircraft accident? ( 5 ) Are these five observations quite unrelated, or do they have a common biochemical basis? As a starting point, it is worthwhile examining the manner in which organisms obtain energy from their food; maybe then we will he able to understand the similarities and subtle differences in the above processes. In simole terms. aerobic life on Earth derives energy - from the oxidation of &gars or fats, for example (6, 7): C,Hz,O,
(Sugar) + nOz
-
nCOa
+ nHzO +Energy
This reart iun is rxt:rgonic. Indeed, a C-6 iugilr such as glurosr libernt(.i ahnut 2800 kJ mul-'. and the redox enercv rr1rast.d is harnessed by an organism to provide energy foractivities such as muscle movement, nerve conduction, and active transport; some of the energy is used to keep the organism warm. The detailed sequence of events underlying the cellular hut it is oxidation of sugars and fats is rather involved (8,9), necessary to note some of the key reactions. During the initial stages, a sugar such as glucose (C6H1206) is split to pyruvic acid (CH3-CO-COOH) which then reacts with coenzyme A(HS-CoA), a rather complex organic molecule (IOa, lob) to vield acetvl-coenzvme A (CHz-CO-S-CoA). an entitv in which . ;he acetyi group is activated for reaction'. ~ c e t ~ l - c o e n z y m e A then enters the tricarboxvlic acid (or Krebs) cycle wherein the acetyl group is compl&ely oxidized to carbon dioxide, coenzyme A is regenerated, and the electrons released reduce either of the two organic redox molecules, nicotinamide adenine dinucleotide, NAD+ ( l l a , l l b ) or flavin adenine dinucleotide, FAD (12a,12b) to NADH or FADHz, respectively; for example:
In actual fact, it is observed that 3 moles of NADH and 1 mole of FADHz are produced via this redox reaction in the tricarboxylic acid cycle. These chemical reductants then donate electrons into an electron transport system (usually called the respiratory redox chain) which conducts them to the ultimate election acceptor, the oxygen (02) molecule, which is reduced to water. It is important to note that a significant amount of the redox free energy released duringelectron
transfer from NADH (or FADHz) to oxygen is harnessed quite efficiently by the electron transport apparatus to generate the "high energy compound" ATP (Adenosine Triphosphate) from ADP (Adenmine Diphosphate) and inorganic phosphate (PO:-) in a process termed "oxidative phosphorylation." We can summarize the overall orocess of oxidative ohosphorylation by the reactions: CeHnOe + 602
38 ADP + 38 PO:-
--
+ E(p)
C6H1206 + 602 + 38 ADP + 38 POI--
6COz + 6Hz0 + E(p) + E(h) 38 ATP
6C02
+ 6Hz0 + 38 ATP
+EM whereE(p) = energy harnessed to produce ATP, andE(h) =energy lost as heat. The overall efficiency of energy capture during this reaction is approximately 40% (13), which is about twice that of the automobile internal combustion engine. Nearly two-thirds of the ATP generated during the oxidation of glucose is obtained via the electron transoort svstem. and hence this biochemical pathway is an important source of energy for an organism. I t is of interest to note that the energy-capturing electron transport apparatus is not unique to aerobic organisms and other electron acceptors can replace oxygen; for example, nitrite and sulfite function as the terminal electron acceptors in nitrite reductase and sulfite reductase, respectively (14). At the cellular level, the extraction of energy via the tricarboxylic acid cycle is achieved by an organelle called the mitochondrion (15a, 15b). This approximately egg-shaped entity has a double membrane structure in which the outer membrane serves to define its shape, while the inner membrane is folded with oortions of it (cristae) projecting into the aqueous interior. ~ h enzymes k necessary for the tri&rboxylic acid cycle are located principally in the internal aqueous cavitv. and the convoluted structure of the inner membrane supports the ancillary electron-transport chain. Since the mitochondrion is the verv site of ATP oroduction. it is aotlv termed the "powerhouse;' of the cell. ~~~
~~
~~~
.
~
0 ,
Figure 1. Route by
which oxygen from the air is delivered to the mitochon-
drion. Volume 59
Number 5 May 1982
389
The route by which oxygen from the air is delivered to the mitochondrion is shown schematically in'Figure 1. In the process, inhaled oxygen is bonded t o the iron ion in themetalloprotein hemoglobin (Hh) contained in the hlood flowing through the lungs; thence, it is delivered to a similar iron metallonrotein mvoelohin (Mh) which acts as an oxveen storagesite locate2 ldjacent to the cells (16-18). In hotGkb and Mh the oxygen hinding site is the iron (11) ion contained in the porphyrin complex (heme) shown in Figure 2(a); the heme prosthetic group is positioned in a cleft in the glohin protein as shown in Figure 2(h), where it is clear that upon oxygenation the 5 coordinate square Dvramidal iron (11) hecomes octahedrally coordinated w h e i it hinds the oxygen molecule. Mh is a monomeric metalloprotein, (illustrated schematically in Figure 2(h)) whereas the Hb molecule is a tetrameric unit, essentially a tetrahedral cluster of four Mb molecules. The four heme groups are identical in Hh, hut there are important differences between the amino acid sequences in the four glohiu proteins. Thus, in contrast to the Mb molecule which can bind onlv une molecule of oxveen. .- , a single Hh molecule has the capacity to hind a maximum of fou;molecules of oxvaen. I t should he noted that Mh eenerallv has a higher affinity for oxygen than Hb, and henceoxygenated Hh flowing in the hlood readily releases its oxygen load to any oxygen deficient Mh it passes, and simultaneously carries away the carhon dioxide produced hv oxidation reactions r difference (respiration) in the tnitgchondrinn. ~ h strurtural twtween .Mb and Hb is signiticant, since the tetrameric nature of Hh enahles it to respond rapidly to differences in oxygen tension; this allows the Hh aggregate to deliver oxygen where i t is needed so that the hardest working cell gets the best supply of oxygen (16). Hh is thus an efficient oxveen-trans~ortmolecule: there are about 250,000 Hb molecul&in a singie red hluud cell (HRC) and about 5.000 million RBC's in eachcrnbfan adult's 5 1 of blood: this provides a plentiful supply of oxygen to areas of the hody such as the brain, heart, and liver which require large amounts of metabolic energy. Heme proteins not only act as oxygen transport (Hb) and storage (Mh) species, hut they also function as redox molecules called cytochromes (19-21). A number of cytochromes have now been identified; they are all based on a heme redox center with the iron ion octahedrally coordinated and acting as a one-electron redox moiety by cycling hetween oxidation states
Fbwe 2. a, Iron pramporphyrin IX (theh e m e g w ) . b, the heme group pasla& in the clefi of the globin protein.
390
Journal of Chemical Education
+2 and +3. The schematic structure of two representative cytochromes is shown below:
The octahedral coordination about each iron ion is emphasized, hut the various suhstituents present on the porohvrin . " rines and the detailed nrotein structures are not shown. The "organic" portion is however important since it "tunes" the redox potential of the iron ion and dictates the solubility characteristics of the cytochrome so that it can function correctlv in the mitochondrial electron-trans~ortchain. he cytochromes, together with seveial organic redox molecules and an ironlcopper metalloprotein, cytochromec-oxidase, constitute the components of the electron-transport chain (2211,226) as illustrated schematicallv in Figure 3, which shows a sugar as the original source of reducing power and oxygen as the final electron acceptor. In addition, Figure 3 indicates that the various redox species operate as ele&rontransport agents hy cycling between their oxidized and reduced forms, with the synthesis of ATP being coupled with some (usually three) of the separate electron-transfer steps. Now, where does this description of cellular energetics have relevance to our initial queries? Carbon monoxide (CO) . . is generated hv the incomolete combustion of carhon compounds in the internal combustion engine of automobiles and also in the smoulder zone of a ciaaritte. Carbon monoxide is similar in size to oxygen, hut both Mb and Hh have a far greater affinity for carhon monoxide than for oxygen (3,23-27). Thus, where there is competition between the redox inactive carhon monoxide and the oxidant oxygen, the tenacious CO readily wins out and the ability of H b to carry oxygen is markedly diminished via the reaction:
Indeed. the K,. value can he used to show that 50%saturation of ~b by 6 is achieved in air containing only 0.1%CO; this corres~ondsto an ap~roximateCO ~ a r t i a Dressure l of 10-3 atm. ~urthermore,i t h a s been repoited (26) that continuous exposure to an atmosphere containing CO a t a partial pressure of 10W atm is sufficient to induce coma! Clearly, the restricted transfer of oxygen through the hody is detrimental, and avictim first lapses into unconsciousness and then a fatal coma unless the effect of carbon monoxide can he overcome. Fortunately, administering oxygen (or air) to the patient is usually sufficient to reverse the facile equilibrium, above. Thus. we can understand whv New York and Tokvo " nolice . take oxygen rest.- to restore the oxygen content of their blood cartxm monoxide from their Hh which to normal bv.disolacinr . has become partial1;saturated with CO from automobile
I 2HP Figure 3. lhe electron transport chain.
exhausts. In addition, carbon monoxide in the hlood of heavy smokers can reduce its oxygen-carrying capacity by up to 20% (23b). Hence, smoking during pregnancy will restrict the supply of oxygen to a developing fetus and so retard its growth. Some plants, especially those grown by "organic" gardeners, have a sap rich in nitrate ion (NO,). Nitrate-reducing bacteria can readily convert nitrate to nitrite (NOp) if the food, particularly silver beet or spinach, is incorrectly stored. The nitrite ion is redox active and can be readily reduced: Thus, the nitrite ion can behave as a one-electron oxidant, a reaction particularly suited to Fe2+-porphyrinspecies which can react as shown below:
nitroso-met-Mb (bright red)
nitrooo-Mb (crimson)
both unaffected by heat (cooking)
myoclobin (purple-red)
cvanide ion is not detectablv bound to Hb in blood (30). Thus. cyanide cannot significantiy interfere with oxygen-delivery to cells but rather must upset the oxypen-utilization reaction that occurs in the mitoch&drion. Asdescribed above (see Fig. 3) the mitochondria1 electron-transport chain contains Fe2+ and Fe3+ in cvtochromes. In addition. the terminal electron-acceptor in the respiratory redox chain, cytochromec-oxidase. although not comwletelv characterized as vet. . . is known to containiwo further'cytochromes denoted a and as, plus two protein-bound copper ions that cycle between Cu2+ and Cu+. These four metal ions are believed to function in concert such that four electrons can be almost simultaneously supplied to oxygen (5)thus: cyt-a(Fe2+),cyt-a3(Fe2+) + eyt-o(FeW),eyt-a3(Fe3+) 2 Cu+ 2Cu2++ 4eO2+ 4Ht + 4e-= 2Hz0 I t is well known that the cyanide ion binds tenaciously to metal ions, particularly Fe3+, and thus the oxidized forms of the cytochromes are likely targets. Current experimental evidence suggests that cytochrome-aa is the primary species affected. Indeed, a concentration of about 10W M CN- is sufficient to completely inhibit this most sensitive cytochrome! (5). Clearly then, the cyanide ion functions by shutting down the process by which mitochondria provide energy for an organism, and if swift action is not taken death can ensue within minutes! Incidentally, some recent research has shown that carbon monoxide can also affect cytochrome-c-oxidase, again prohably by ligand (CO) coordination to cytochrome-as. (23a, 32, 33). Thus. carbon monoxide has a doublv sinister effect on mi'tochondrial energy production: not onl; does it restrict the access of oxveen to the res~iratorvredox chain. but also it can inhibit the electron-transfer step in whichoxygen is reduced. The antidote for cyanide poisoning is an intravenous injection of a solution-containing sodium nitrite (NaN02)and sodium thiosulfate (NazSzOs) together with the inhalation of oxygen, if possible (.5). We can understand the purpose of each component on the basis of our previous discussions. As wehave seen, the presence of the nitrite ion in hlood leads to the formation of met-Hh (Hb containing Few) and met-Hb-NO, both of which readilv take UD CN- to form met-Hb-CN. Thus.. cvanide ion . circulating in the blood stream is rapidly sequestered by oxidized Hb and to compensate fur the destruction of a small proportion of the hlood Hb, oxygen is administered to ensure a sufficient supply is maintained to the cells. But what about the cyanide that has penetrated the cells and what role does the NazSzOn ~ l .a in v the antidote? T o an- ~. swer these questions we must realize that the mammalian biosystem has several built-in mechanisms for detoxifying intracellular cyanide: the major route converts it to the relatively harmless thiocyanate ion (-SCN) via the enzymic reaction:
"-
met-Mb (brown)
henkin (brown)
The reaction with the nitrite ion can have catastrophic vonsequenws since it inactivates both hlb and Hh I,y, ti) oxidiping them t o the Fe"+ forms, met-1lt1 and met-Hh (the "met" prefix denotes r t ~ eirun is present as F'e"', which have no uxygen-carrier nvtivity, and ( i i ) blurking the oaygm r m r dination site with the mure firmly hnmd lirnnd KO.12dJ. In babies and infants the consumption of ~ 0 , / ~ 0 p c o n t a i n i n g foods can thus create an oxygen-deficiency crisis with the clinical name methemoglobinemia (3,291. This condition can be fatal in infants due to their lower hlood volume and higher metabolic demands than adults. Clearly, the "pumping" of meat is based on the above discussion ( 4 ) . The nitrite ion Dresent in brine can aaain oxidize the Fez+-porphyrinin hlh,ihusfurming the red iron-nitroso porphyrin deri\.atives. Hence, the meat maintains a hrighr red appearance during couking instead ot i t color deteriorating toa dull hruwn a s a result of the pr~rductionofaquo-met-Mb specie3 when hlh iiaeruhicnlly oxidi~edand theglol~inp m r m thermally denatured during cooking (see above). Polvacrvlonitrile materials such as Orlon" or Acrilan" and cyanopolymer plastics are used in aircraft furnishings. Unfortuuatelv. .. all these materials can "give rise to smokes containing hydrogen cyanide (HCN)-up to 1.5% w/w-when burned in air (5). Upon inhalation, HCN is readily absorbed via the lungs into the circulatory system. However, contrary to popular belief its major target is not H b or Mb! (23a, 30). This is a common misconception and is even perpetrated in several well-known textbooks (25, 27, 311, probably because the cyanide ion (CN-) is similar in size and isoelectronic with carbon munoxide. Nevertheless, experimental evidence indicates that the
. -
CN-
+ Sulfur substrate
Sulfur-trsnsrersse
-SCN
IRhodaneasl
In mammals, the supply of enzyme is not the limiting factor. Indeed, a dog contains enough sulfur-trausferase to convert about 4 kg of CN- to SCN- in 15 minutes! (5) Clearly, the availability of a suitable sulfur-containing substrate, such as cysteine, cystine, or glutathione, is the rate-determiningfactor. In the case of the cyanide antidote, i t is obvious that the function of the NazSzOs is to provide an adequate supply of the good sulfur-substrate SzOi-to the mitochondria so that detoxification can proceed readily. Thus, we can now appreciate why heme metalloprotein complexes are essential in the delivery and utilization of Oz in cellular biochemistry. Moreover, we should note that this delicate machinery can be partially or completely suppressed by quite simple chemical species such as CO, NO;, or CN-. Volume 59
Number 5 May 1982
39 1
Literature Clted ( I ) Drummond, &.,A. H.,Seiqusst,53,20(1980). (2) MacMahon, B., Alpert, M., and Salber, E. J., A m r . J. Epidemiology, 82, 247 il(lR5,
(3) R&S: F., and Soratnkke, R. B., J. CHEM. EDUC.,50,347 (1973). (4) Lawie. R. A.."Mest Seicnee." 2nd. Ed..Pergamon Prear, Oxford, 1974, pp. 259.262, 290. (5) Labianea, D. A,. J. CDM. EDUC.,56,788 (1979). (6) Edwards. N. A,, and HasmII. K. A,, "Cellula. Biochemistry and Physiology: McGraw-Hill, London, 1971. p. 137. (7) Conn.E. E.,andStumpf.P. K.."OutlinesofBIahemiatry."4th Ed.. John Wiley&Sans, he., New York, 1976,pp.328.329. (8) Ref (6). Chapter 7. pp. 159-174. (9) Ref. (7). Chapter 12, pp. 327-344. (10) (a) Ref. (61.p.88: (b) Rci. (7j.p. 227. (11) (a) Ref. (6),p.84: (b) Ref. (71.p. 199. ~ R e t (7b.p. 205. (12) la) R e t ( 6 1 , l461b) (13) Ref. 17). p. 395. (14) Ochisi,E.,"Bioina~anieChemistry,AnInhoduetion."AUynandBaeon,B~toto,1977, pp. 454,458. (15) (a) R e t (6). pp. 2628: (b) Ref. (7). pp. 262265. (16) Peru*, M. F., '"Hemoglobin Structure and Respiratory Transport." Sei. Amer, 239, 68 (1978).
392
Journal of Chemical Education
F I r 8 m r 8 hl Gmrdmna. B and llann.ifrr..l V ..n I n ~ ~ a A 0 ,* b d t h % T h < I I&
