The effects of mutagenesis on geminate and bimolecular O 2 rebinding to 90 mutants at 27 different positions were used to map pathways for ligand movement into and out of sperm whale myoglobin. By analogy to a baseball glove, the protein "catches" and then "holds"

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Geminate oxygen rebinding to myoglobin was followed from a few nanoseconds to a few microseconds after photolysis for more than 25 different oxymyoglobin point mutants in the presence and absence of 12 atm of xenon. In all cases, two relaxations were observed: an initial fast phase (half-time 20 ns) and a slower, smaller phase (half-time 0.5-2 micros). Generally, xenon accelerates the fast reaction but slows the slower reaction and diminishes its amplitude. The rates and proportions of the two components and the effects of xenon on them vary widely for different mutants. The locations of specific xenon binding sites [Tilton, R. F., Kuntz, I. D. Jr., and Petsko, G. A. (1984) Biochemistry 23, 2849-2857], the effects of point mutations on the geminate reactions, and molecular dynamics simulations were used to suggest locations in the protein interior occupied by ligands on the nanosecond to microsecond time scale. Photodissociated ligands may occupy xenon site 4 in the distal pocket and xenon site 1 below the plane of the heme. Rebinding from these positions corresponds to the slower geminate phase for O2 rebinding. The rapid geminate component is determined by competition between rebinding from a position closer to the iron atom and escape to solvent or more distant locations in the protein.

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One of the most remarkable structural aspects of Scapharca dimeric hemoglobin is the disruption of a very well-ordered water cluster at the subunit interface upon ligand binding. We have explored the role of these crystallographically observed water molecules by site-directed mutagenesis and osmotic stress techniques. The isosteric mutation of Thr-72 3 Val in the interface increases oxygen affinity more than 40-fold with a surprising enhancement of cooperativity. The only significant structural effect of this mutation is to destabilize two ordered water molecules in the deoxy interface. Wild-type Scapharca hemoglobin is strongly sensitive to osmotic conditions. Upon addition of glycerol, striking changes in Raman spectrum of the deoxy form are observed that indicate a transition toward the liganded form. Increased osmotic pressure, which lowers the oxygen affinity in human hemoglobin, raises the oxygen affinity of Scapharca hemoglobin regardless of whether the solute is glycerol, glucose, or sucrose. Analysis of these results provides an estimate of six water molecules lost upon oxygen binding to the dimer, in good agreement with eight predicted from crystal structures. These experiments suggest that the observed cluster of interfacial water molecules plays a crucial role in communication between subunits.Water plays a unique and ubiquitous role in biology. Folding, stability, and function of protein molecules are all strongly influenced by their interactions with water molecules. Despite the importance of these interactions, the precise role of water in structure and function is often difficult to elucidate. Ordered water molecules are nearly universally observed in macromolecular crystallographic analysis (1, 2), and biologically important roles for these observed water molecules have been implicated in a few cases including substrate specificity (3), catalysis (4), antigen-antibody association (5), and DNA recognition (6).High resolution x-ray crystallography on the dimeric hemoglobin (HbI) from Scapharca inaequivalvis has revealed a striking ligand-linked rearrangement of water molecules at the subunit interface (7,8). HbI binds oxygen cooperatively with a Hill coefficient of 1.5 and shows no change in oxygen affinity or cooperativity as pH varies from 5.5 to 9.0 (9). Quite unlike mammalian hemoglobins, where cooperativity is mediated by large quaternary structural changes (10), HbI displays minimal ligand-linked quaternary changes. The oxygen affinity of each subunit appears to depend largely on the position of the side chain of Phe-97 which packs in the heme pocket in the deoxy state, but upon heme ligation is extruded into the subunit interface where it expels several water molecules (see Fig. 1). The core of the deoxy interface includes a cluster of 17 very well-ordered water molecules, 6 of which are lost upon oxygenation. (An additional two water molecules within the heme pocket are directly displaced by the bound oxygen.)We have undertaken the present studies to determine the functional import...

During the past 50 years an enormous amount of work has been done on the equilibria and the kinetics of the reversible reaction of mammalian haemoglobin with oxygen. Such work has not only been of obvious importance in respiratory physiology, but has also helped greatly towards the elucidation of the detailed physico-chemical mechanism of this vitally important reaction. The kinetics and equilibria of the reactions of haemoglobin with carbon monoxide have likewise been studied in much detail, not only because their close physico-chemical analogies with the oxygen-haemoglobin reactions might well throw further light on the latter, but also because of their importance in the understanding of (a) the poisonous action of carbon monoxide, and (b) the use of carbon monoxide as a physiological tool, especially in the determination of the diffusing capacity of the human lung and of the average time spent by the blood in the lung capillaries (see, for example, Roughton, 1945).There are a few other substances which combine reversibly with reduced haemoglobin in a similar way to oxygen and carbon monoxide. Of these the best-known are nitric oxide and the isocyanides. St George & Pauling (1951) have studied the effect on the affinity for haemoglobin of varying the size of the group to which the isocyanide radical is attached, and from their results have inferred that the iron centres in haemoglobin are buried in crevices, a view which has been strongly controverted by Keilin (1953). Little or no quantitative work seems, however, to have been done on the physical chemistry of the reaction of haemoglobin with nitric oxide since Hermann's discovery of nitric oxide haemoglobin almost a century ago. Hermann (1865) found, inter alia, that nitric oxide has at least five times greater affinity than carbon monoxide for haemoglobin, and accordingly is able to displace CO readily from combination with haemoglobin. His careful gasometric experiments showed that the volume of carbon monoxide displaceable from fully saturated carboxyhaemoglobin is equal to the volume of nitric oxide absorbed by the haemoglobin, and