Science/Technology are linked by an overlying atmo sphere and an underlying deep sea. One column represents the Antarc tic region and the other the nonAntarctic region of the ocean. Because the fate of the upwelled water in the Antarctic is not precise ly known, Peng and Broecker stud ied two limiting cases. In the first, the water is transferred entirely to the surface of the non-Antarctic col umn. This is the least favorable sce nario for an iron fertilization pro gram because much of the C 0 2 ab sorbed by Antarctic Ocean waters would reenter the atmosphere when it reached the temperate region. In the second case, the water is trans ferred entirely to the deep reservoir. This is the most favorable scenario for iron fertilization because the ab sorbed C 0 2 would no longer be in contact with the atmosphere. The point is it doesn't make much difference which process dominates. In neither case does iron fertilization significantly reduce atmospheric C0 2 . In the first case, after 100 years, atmospheric C 0 2 is reduced only 15 ppm. In the second case, atmospheric C 0 2 is reduced about 35 ppm. Cur rently, the C 0 2 content of the atmo sphere is about 350 ppm. It is project ed to rise 200 to 300 ppm over the next 100 years if present patterns of energy use persist. Broecker says the dynamics of this process tend to confuse biologists. Atmospheric C 0 2 will flow into the Antarctic Ocean only in response to a drop in p c o , in the ocean water. An iron fertilization program in the Ant arctic would result in an initial, sharp drop in ρ COo in offshore waters, and atmospheric C 0 2 would be reduced as C 0 2 flowed into these waters. However, after a few years, the sur face Pco, would reach a near-con stant disequilibrium with the atmo sphere and further decreases in at mospheric C 0 2 would be quite slow. "This is an enormously interesting phenomenon to look into/' Broecker says. However, proponents of an iron fertilization program have suggested it could result in the removal of huge quantities of carbon dioxide, and, Broecker says, "in our model, we don't get a very large effect." Martin says the upwelling rate cal culated by Broecker and Peng is "quite low," and that upwelling rates 30
February 4, 1991 C&EN
calculated by other modelers on the basis of species other than 14C in ocean waters are significantly higher. "I still have tremendous faith in the power of iron," Martin says. "I think we need to design a large-scale iron
enrichment experiment, let the modelers predict the effect on atmospheric C0 2 , and then go out and actually do it. That way we can determine which models are accurate." Rudy Baum
New studies pinpoint pathway of B 12 biosynthesis Teams in France and the U.K. have pooled their expertise to shed new light on the mechanistic details in volved in the biosynthesis of vita min B12. The isolation in France, and identification of an intermediate called precorrin-6x, unknown until now, has changed the entire direc tion of research on the vitamin, maintains Cambridge University's Alan R. Battersby. The finding stems from studies at the Vitry research center of France's Rhône-Poulenc, the largest commercial producer of vitamin B12 in the world. Francis Blanche, Laurent Debussche, and Denis Thibaut at the
center's analytical chemistry department noticed that an unusual material, which proved to be precorrin6x, is formed when a reducing agent is absent from the incubation medium. Surprisingly, despite many decades of study of the vitamin and of methods for its production by researchers around the world, this is the first evidence that vitamin B12's biosynthetic pathway requires the i n t e r v e n t i o n of external redox agents. Until now, it was generally believed such agents weren't necessary. Nuclear magnetic resonance spectroscopy studies by the French
Vitamin B12: a most formidable biosynthetic problem Vitamin B 1 2 , the antiperniciousanemia factor, is among the most complex of the naturally occurring compounds. Known less commonly as cyanocobalamin, it consists of a porphyrinlike ring system in which two of the four substituted pyrrole rings are connected directly with one another rather than through a methine bridging group. Ribofuranose links one of these pyrrole-derived rings with 5,6-dimethylbenzimidazoie. One of the latter's nitrogen atoms and those of the substituted pyrrole rings are complexed with trivalent cobalt
H2NOC CONH2 H2NOC H3CV
CONH 2
H2NOC
Vitamin Β-)2
Numerous scientists around the world, many of them Nobel Laureates, have been working on various aspects of vitamin B 12 for many years. Switzerland's Albert Eschenmoser, x-ray crystallographer Dorothy C. Hodgkin and Lord Todd of the U.K., and the late Robert B. Woodward are among those who have made major contributions in determining this vitamin's chemistry and structure. And studies continue toward understanding details of its formation, which has been referred to as "the Everest of biosynthetic problems." These studies have fo cused largely on the corrinoid macrocycle segment of the vitamin, cobyrinic acid. Biosynthesis starts with two molecules of 5-aminolevulinic acid interacting to yield porphobilinogen (PBG), a pyrrole ring with an aminomethyl group on C-2, an acetate on C-3, and a propionate on C-4. Four PBGs link together to form hydroxymethylbilane, which in turn cyclizes. One unusual feature of the result-
team, and by Battersby and Finian J. Leeper at Cambridge, provide a detailed atom-by-atom picture of the way the vitamin's complex macrocyclic system is built up [Proc. Natl. Acad. Sci., 87, 8795 and 8800 (1990)]. A cell-free extract derived from a special strain of Pseudomonas denitrificans was employed in the latest experiments. The organism, which Rhône-Poulenc uses in its commercial production of vitamin B12, was genetically modified for the study by Joel Crouzet's team at the company to enhance expression of eight of the genes that determine the biosynthesis of the vitamin. Key to the results was an elegant series of experiments starting with v i t a m i n B 1 2 's initial p r e c u r s o r , 5-aminolevulinic acid tagged at various positions along its chain with 13 C, 14 C, and 3 H, together with Relabeled S-adenosylmethionine, a naturally occurring methylating agent. In addition, a pulse-labeling technique that Battersby and his
Cambridge coworkers had developed earlier was employed. Based on use of 13C in conjunction with two-dimensional NMR spectroscopy, the data recorded allow accurate determination of the detailed positions on the macrocyclic molecule that entering methyl groups occupy, as well as the order of each entry. Prior to the discovery of precorrin-6x, it already had been established that the early stages in vitamin B12's macrocycle biosynthesis involve stepwise methylation of uroporphyrinogen III, first at position 2, then 7, followed by 20. The trimethylated intermediate is precorrin-3. The French and British chemists now have proved the methylations continue—at carbons 17, 11 (rather than 12 as previously thought), and 1 in that order— accompanied by elimination of the bridging methine group at position 20 as acetic acid. That loss results in contraction of the macrocycle ring size by one carbon atom.
Ing uroporphyrinogen III Is that the acetate and propionate substltuents on one ring are In the reverse order from those of the other three rings
H 2 N— C H 2 — C — C H 2 —CH 2 —COOH
At this stage, biosynthesis stops if reduced nicotinamide adenine dinucleotide phosphate (NADPH), a naturally o c c u r r i n g cofactor, i s n ' t present. The resulting precorrin-6x, which has been isolated and purified, is a light yellow-colored material. Subsequent addition of NADPH to the incubation mixture brings about reduction of precorrin-6x, followed by two events. One is continuation of the methylations, at positions 5 and 15. The other event, surprising from a mechanistic point of view, centers on C-12. There, the acetate moiety loses carbon dioxide, resulting in the eighth and final methyl group on the macrocyclic ring. At this stage also, the methyl group on the neighboring C-ll migrates to C-12. The product, h y d r o g e n o b y r i n i c acid, is the cobalt-free form of cobyrinic acid, a late precursor of vitamin B12. Dermot O'Sullivan
and heme. The other results in vitamin B 12 through a series of methylated Intermediates—first precorrin-1, then precorrln-2, followed by precorrln-3, and on through some current ly unknown steps to precorrln-6x. Then comes another gap in current knowledge before the cobalt-free hydrogenoby rinic acid Is reached and, ultimately, cobalt-containing cobyrlnic acid
5-Amlnolevullnlc acid Uroporphyrinogen III
R
Λ
K
R
A
K
R
K A
ΪΧ JfX
X\ i t Η
R2
RK
Η
Η
R2
»H
H3C
*>Ri
FV.H HHsC! A
Η
Hydroxymethylbilane
Pi Heme
H2C
CH 2
VNH
Vitamin B12
HN^(
Chlorophyll R2
n
2
Uroporphyrinogen III t1 = -CH2-COOH
R2 = -CH2-CH2-COOH
From uroporphyrinogen III, enzyme-mediated reactions fol low two broad avenues. One leads ultimately to chlorophyll
February 4, 1991 C&EN
31
