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Paper #2 uncovers for the first time the way the trick is performed, or at least how a similar enzyme, chloroperoxidase, carries it out. This enzyme is a strong enough oxidizing agent to be able to add an oxygen atom to a relatively unreactive molecule and yet not to oxidize the chemically more vulnerable protein structure of which the enzyme itself is made. So what is its secret? Michael T. Green at the Department of Chemistry at Pennsylvania State University, John Dawson at the University of South Carolina, and Harry B. Gray of the Beckman Institute at Caltech, used EXAFS (extended X-ray absorption fine structure) spectroscopy to show what happens. The answer lies in the nature of the ferryl bond itself. The ferryl form of chloroperoxidase (known as CPO-II) was the only ferryl species for which an FeIV=O stretching mode could not be found in the vibrational spectrum, and this would normally be in the region of 655-875 cm-1. Even replacing the O atom with the heavier 18O isotope, which would cause a detectable shift in frequency, failed to identify it. Green and co-workers found that the Fe–O bond length in CPO-II is 1.82 Å rather than the 1.65 Å typical of a ferryl species. Consequently they predicted that the ferryl unit in CPO-II was protonated. This suggestion was somewhat controversial, in that no synthetic or mineral FeIV–OH had ever been characterized.More recent work by Green, using Mössbauer and resonance Raman spectroscopy, confirms that their predictions were right as shown by the Fe–OH stretching frequency which should be around 563 cm-1. By making resonance Raman measurements of CPO-II and of its isotopically substituted versions with 18O and 2H atoms, Green has proved the existence of an FeIV–OH in this enzyme (see K.L. Stone, et al., PNAS, 103[33]: 12307-10, 2006). More recently he and his co-workers have published results for cytochrome P450 (see R.K. Behan, et al., J. Am. Chem. Soc., 128[35]: 11471-4, 2006). The change in behavior of the ferryl group is attributed to strong electron donation from the other side of the iron thanks to the sulfur atom of a thiolate ligand. Nature appears to use thiolate-ligated hemes to perform these hydroxylation reactions. The enzymes cytochrome P450, nitric oxide synthase, and chloroperoxidase are the only heme systems known to hydroxylate substrates, and these have a thiolate group as ligand to the iron. Green believes that the basic ferryls afforded by thiolate-ligation may promote the hydroxylation process, and his group is examining if basic ferryl species are indeed a general and unique feature of thiolate-ligated hemes. Speaking to Science Watch, Green explains why his research has gained such a high position in the Hot Ten list: "Thiolate-ligated heme-proteins are an important class of metabolic enzymes. The aim of our studies is a better understanding of the electronic and geometrical structures of the high-valent intermediates found in their catalytic cycles. Knowledge gained from our studies could be parlayed into improved catalysts for industrial applications. The enzymes we study use only electrons, protons, and O2 (or peroxide) to oxidize substrates, and the only by-product is water. These enzymes really are ‘green’ catalysts, and synthetic systems that could mimic them would be of significant value." Will Green one day be among the vanguard of the green revolution? It seems more than likely. 
Dr. John Emsley is based at the Department of Chemistry,
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ron(IV)-oxygen
(ferryl) bonds are a crucial part of important enzymes such as the methane monooxygenase which converts methane to methanol, the ribonucleotide reductase which constructs the nucleotides for DNA, and the HIF prolyl hydroxylase which regulates the level of oxygen in tissue, ensuring it remains at an optimum level. There is also a ferryl unit at the heart of cytochrome P450 which metabolizes pharmaceuticals as well as removing unwanted chemicals from the body. P450 can convert C–H bonds to C–OH bonds, thereby solubilizing a molecule sufficiently for it to be discharged via the urine. The ferryl group abstracts a hydrogen from a substrate and then returns it as an OH group so that C–H becomes C–OH and the properties of the substrate are radically changed from hydrophobic to hydrophilic.
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