Life on earth essentially depends on the functioning of biological macromolecules such as proteins. At least 50 % of all known enzymes contain metal atoms as key constituents of their active sites, which are crucially for the reactivity. These metal centers often are chemically complex and involved in electron and proton transfer processes, substrate turnover, and regulation. A particular important aspect in biophysics research is the activation of small molecules at protein-bound metal centers with outstanding relevance in chemistry, medicine, industry, and energy applications. The field is still under rapid development. The lecture is addressed to advanced students with interests in biophysics. Covered topics include, e.g., coordination chemistry, assembly, molecular and electronic structure, photoreactions, redox processes, catalysis, and dynamics of metal centers in, for example, hydrogenase, photosystem, oxidase proteins. Selected experimental and theoretical approaches to understand the mechanisms, with emphasis on X-ray based techniques such as spectroscopy and crystallography, are introduced. Recent scientific highlights in molecular biophysics are discussed. The aim is to provide an introduction into important questions, practical approaches, and outcomes of biophysical research on metalloenzymes.